Rahman RN Zaliha Raja Abdul RahmanEnzyme and Microbial Technology Research, Faculty of Science and Environmental Studies, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor MalaysiaPhone : Fax :
The regioselectivity characteristic of lipases facilitate a wide range of novel molecule unit constructions and fat modifications. Lipases can be categorized as sn-1,3, sn-2, and random regiospecific. Geobacillus zalihae T1 lipase catalyzes the hydrolysis of the sn-1,3 acylglycerol chain. The T1 lipase structural analysis shows that the oxyanion hole F16 and its lid domain undergo structural rearrangement upon activation. Site-directed mutagenesis was performed by substituting the lid domain residues (F180G and F181S) and the oxyanion hole residue (F16W) in order to study their effects on the structural changes and regioselectivity. The novel lipase mutant 3M switches the regioselectivity from sn-1,3 to only sn-3. The mutant 3M shifts the optimum pH to 10, alters selectivity toward p-nitrophenyl ester selectivity to C14-C18, and maintains a similar catalytic efficiency of 518.4 x 10-6 (s-1/mM). The secondary structure of 3M lipase comprises 15.8% and 26.3% of the alpha-helix and beta-sheet, respectively, with a predicted melting temperature (Tm) value of 67.8 C. The in silico analysis was conducted to reveal the structural changes caused by the F180G/F181S/F16W mutations in blocking the binding of the sn-1 acylglycerol chain and orientating the substrate to bond to the sn-3 acylglycerol, which resulted in switching the T1 lipase regioselectivity.
Determining structure of highly flexible protein with multiple conformations can be challenging. This paper aims to combine molecular dynamics (MD) and small angle X-ray diffraction (SAX) techniques as a solution to overcome issues related to protein conformation in hardly crystallized protein. Based on prior studies, a cold-active lipase AMS8 was simulated in solvents showing stability in its N-terminal and high flexibility in its C-terminal. However, MD in its own algorithm could not explain the basis of macromolecule conformational transitions or changes related to protein through folding. Hence, by combining SAXS with MD, it is possible to understand the structure of flexible AMS8 lipase in natural space. Based on the findings, SAXS ab-initio model of AMS8 lipase was identified as a monomeric protein in which the optimized model of cold-active lipase AMS8 derived from SAXS data was found to be aligned with AMS8 homology model under series of MD timeframe.
5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 A with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl-esters.
Title: An integrated overview of bacterial carboxylesterase: Structure, function and biocatalytic applications Johan UUM, Rahman RNZRA, Kamarudin NHA, Ali MSM Ref: Colloids Surf B Biointerfaces, 205:111882, 2021 : PubMed
Carboxylesterases (CEs) are members of prominent esterase, and as their name imply, they catalyze the cleavage of ester linkages. By far, a considerable number of novel CEs have been identified to investigate their exquisite physiological and biochemical properties. They are abundant enzymes in nature, widely distributed in relatively broad temperature range and in various sources; both macroorganisms and microorganisms. Given the importance of these enzymes in broad industries, interest in the study of their mechanisms and structural-based engineering are greatly increasing. This review presents the current state of knowledge and understanding about the structure and functions of this ester-metabolizing enzyme, primarily from bacterial sources. In addition, the potential biotechnological applications of bacterial CEs are also encompassed. This review will be useful in understanding the molecular basis and structural protein of bacterial CEs that are significant for the advancement of enzymology field in industries.
Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The k(m) and k(cat) values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.
Title: Heterologous Expression and Characterization of Plant Lipase LIP2 from Elaeis guineensis Jacq. Oil Palm Mesocarp in Escherichia coli Mohd Din MH, Nair A, Masomian M, Ali MSM, Rahman RNZRA Ref: Catalysts, 11:244, 2021 : PubMed
In order to determine the potential of biochemical and structural features of Elaeis guineensis Jacq. oil palm mesocarp lipases, the LIP2 gene was isolated, expressed, purified and characterized through the Escherichia coli microbial recombinant system. Gene analysis of LIP2 revealed that it is composed of 1584 base pairs which are encoded in 528 amino acid residues with a molecular weight of around 57 kDa. LIP2 has distinctive lipolytic properties in terms of alpha/beta fold and the catalytic triad for lipase. The LIP2 lipase was successfully expressed and purified from E. coli Rosetta (DE3) via affinity chromatography. The optimal temperature and pH for the lipase activity was 30 degC and a pH of 9, respectively. Stability was profoundly increased with the addition of metal ions (Ca2+, Mg2+, Mn+, and Ni+), along with organic solvents (ethanol and octanol). pNP myristate was the most suitable among all pNP esters. In biophysical characterization analysis, LIP2 has a thermal denaturing point at 66 degC, which mostly consists of random patterns (39.8%) followed by alpha-helix (30.3%), turns (23.8%) and beta-sheet (6.2%). From the successful purification and characterization, the potential of oil palm mesocarp lipase was able to be further explored.
Title: Extraction and reimmobilization of used commercial lipase from industrial waste Mokhtar NF, Rahman RNZRA, Sani F, Ali MSM Ref: Int J Biol Macromol, :, 2021 : PubMed
In industrial application, immobilized lipase are typically not reused and served as industrial waste after a certain process is completed. The capacity on the reusability of the spent lipase is not well studied. This current study embarks on reusing the remaining lipase from the spent immobilized enzyme. Active lipases were recovered using a simple reverse micellar extraction (RME). RME is the extraction process of targeted biomolecules using an organic solvent and a surfactant. This method was the first attempt reported on the recovery of the lipase from the used immobilized lipase. RME of the spent lipase was done using the nonionic Triton X-100 surfactant and toluene. Various parameters were optimized to maximize the lipase recovery from the used immobilized lipase. The optimum forward extraction condition was 0.075 M KCl, and backward conditions were at 0.15 M Triton X-100/toluene (pH 6, 2 M KCl) with recovery of 66%. The extracted lipase was immobilized via simple adsorption into the ethanol pretreated carrier. The optimum conditions of immobilization resulted in 96% of the extracted lipase was reimmobilized. The reimmobilized lipase was incubated for 20 h in pH 6 buffer at 50 degreesC of water bath shaker. The reimmobilized lipase still had 27% residual activity after 18 h of incubation, which higher thermal stability compared to the free lipase. In conclusion, the free lipase was successfully extracted from the spent immobilized lipase and reimmobilized into the new support. It exhibited high thermal stability, and the reusability of the spent lipase will promote continued use of industrial lipase and reduce the cost of the manufacturing process.
Title: Ancestral Sequence Reconstruction of Ancient Lipase from Family I.3 Bacterial Lipolytic Enzymes Rozi MFAM, Rahman RNZRA, Leow ATC, Ali MSM Ref: Mol Phylogenet Evol, :107381, 2021 : PubMed
Family I.3 lipase is distinguished from other families by the amino acid sequence and secretion mechanism. Little is known about the evolutionary process driving these differences. This study attempt to understand how the diverse temperature stabilities of bacterial lipases from family I.3 evolved. To achieve that, eighty-three protein sequences sharing a minimum 30% sequence identity with Antarctic Pseudomonas sp. AMS8 lipase were used to infer phylogenetic tree. Using ancestral sequence reconstruction (ASR) technique, the last universal common ancestor (LUCA) sequence of family I.3 was reconstructed. A gene encoding LUCA was synthesized, cloned and expressed as inclusion bodies in E. coli system. Insoluble form of LUCA was refolded using urea dilution method and then purified using affinity chromatography. The purified LUCA exhibited an optimum temperature and pH at 70 degC and 10 respectively. Various metal ions increased or retained the activity of LUCA. LUCA also demonstrated tolerance towards various organic solvents in 25% v/v concentration. The finding from this study could support the understanding on temperature and environment during ancient time. In overall, reconstructed ancestral enzymes have improved physicochemical properties that make them suitable for industrial applications and ASR technique can be employed as a general technique for enzyme engineering.
Title: Microbial Biodegradation of Paraffin Wax in Malaysian Crude Oil Mediated by Degradative Enzymes Adlan NA, Sabri S, Masomian M, Ali MSM, Rahman RNZRA Ref: Front Microbiol, 11:565608, 2020 : PubMed
The deposition of paraffin wax in crude oil is a problem faced by the oil and gas industry during extraction, transportation, and refining of crude oil. Most of the commercialized chemical additives to prevent wax are expensive and toxic. As an environmentally friendly alternative, this study aims to find a novel thermophilic bacterial strain capable of degrading paraffin wax in crude oil to control wax deposition. To achieve this, the biodegradation of crude oil paraffin wax by 11 bacteria isolated from seawater and oil-contaminated soil samples was investigated at 70degC. The bacteria were identified as Geobacillus kaustophilus N3A7, NFA23, DFY1, Geobacillus jurassicus MK7, Geobacillus thermocatenulatus T7, Parageobacillus caldoxylosilyticus DFY3 and AZ72, Anoxybacillus geothermalis D9, Geobacillus stearothermophilus SA36, AD11, and AD24. The GCMS analysis showed that strains N3A7, MK7, DFY1, AD11, and AD24 achieved more than 70% biodegradation efficiency of crude oil in a short period (3 days). Notably, most of the strains could completely degrade C(37)-C(40) and increase the ratio of C(14)-C(18), especially during the initial 2 days incubation. In addition, the degradation of crude oil also resulted in changes in the pH of the medium. The degradation of crude oil is associated with the production of degradative enzymes such as alkane monooxygenase, alcohol dehydrogenase, lipase, and esterase. Among the 11 strains, the highest activities of alkane monooxygenase were recorded in strain AD24. A comparatively higher overall alcohol dehydrogenase, lipase, and esterase activities were observed in strains N3A7, MK7, DFY1, AD11, and AD24. Thus, there is a potential to use these strains in oil reservoirs, crude oil processing, and recovery to control wax deposition. Their ability to withstand high temperature and produce degradative enzymes for long-chain hydrocarbon degradation led to an increase in the short-chain hydrocarbon ratio, and subsequently, improving the quality of the oil.
Title: The Role of Surface Exposed Lysine in Conformational Stability and Functional Properties of Lipase from Staphylococcus Family Ahmad NN, Kamarudin NHA, Leow ATC, Rahman RNZRA Ref: Molecules, 25:, 2020 : PubMed
Surface charge residues have been recognized as one of the stability determinants in protein. In this study, we sought to compare and analyse the stability and conformational dynamics of staphylococcal lipase mutants with surface lysine mutation using computational and experimental methods. Three highly mutable and exposed lysine residues (Lys91, Lys177, Lys325) were targeted to generate six mutant lipases in silico. The model structures were simulated in water environment at 25 degC. Our simulations showed that the stability was compromised when Lys177 was substituted while mutation at position 91 and 325 improved the stability. To illustrate the putative alterations of enzyme stability in the stabilising mutants, we characterized single mutant K325G and double mutant K91A/K325G. Both mutants showed a 5 degC change in optimal temperature compared to their wild type. Single mutant K325G rendered a longer half-life at 25 degC (T(1/2) = 21 h) while double mutant K91A/K325G retained only 40% of relative activity after 12 h incubation. The optimal pH for mutant K325G was shifted from 8 to 9 and similar substrate preference was observed for the wild type and two mutants. Our findings indicate that surface lysine mutation alters the enzymatic behaviour and, thus, rationalizes the functional effects of surface exposed lysine in conformational stability and activity of this lipase.
Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.
Title: Calcium-Induced Activity and Folding of a Repeat in Toxin Lipase from Antarctic Pseudomonas fluorescens Strain AMS8 Ali NSM, Salleh AB, Rahman RNZRA, Leow ATC, Ali MSM Ref: Toxins (Basel), 12:27, 2020 : PubMed
It is hypothesized that the Ca(2+) ions were involved in the activity, folding and stabilization of many protein structures. Many of these proteins contain repeat in toxin (RTX) motifs. AMS8 lipase from Antarctic Pseudomonas fluorescens strain AMS8 was found to have three RTX motifs. So, this research aimed to examine the influence of Ca(2+) ion towards the activity and folding of AMS8 lipase through various biophysical characterizations. The results showed that CaCl2 increased lipase activity. The far-UV circular dichroism (CD) and Fourier-transform infrared (FTIR) analysis suggested that the secondary structure content was improved with the addition of CaCl2. Fluorescence spectroscopy analysis showed that the presence of CaCl2 increased protein folding and compactness. Dynamic light scattering (DLS) analysis suggested that AMS8 lipase became aggregated at a high concentration of CaCl2.The binding constant (Kd) value from the isothermal titration calorimetry (ITC) analysis proved that the Ca(2+) ion was tightly bound to the AMS8 lipase. In conclusion, Ca(2+) ions play crucial roles in the activity and folding of the AMS8 lipase. Calcium binding to RTX nonapeptide repeats sequences will induced the formation and folding of the RTX parallel beta-roll motif repeat structure.
Title: The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8 Ali NSM, Salleh AB, Leow ATC, Rahman RNZRA, Ali MSM Ref: Toxins (Basel), 12:579, 2020 : PubMed
Calcium-binding plays a decisive role in the folding and stabilization of many RTX proteins, especially for the RTX domain. Although many studies have been conducted to prove the contribution of Ca(2+) ion toward the folding and stabilization of RTX proteins, its functional dynamics and conformational structural changes remain elusive. Here, molecular docking and molecular dynamics (MD) simulations were performed to analyze the contribution of Ca(2+) ion toward the folding and stabilization of the RTX lipase (AMS8 lipase) structure. AMS8 lipase contains six Ca(2+) ions (Ca1-Ca6). Three Ca(2+) ions (Ca3, Ca4, and Ca5) were bound to the RTX parallel beta-roll motif repeat structure (RTX domain). The metal ion (Ca(2+)) docking analysis gives a high binding energy, especially for Ca4 and Ca5 which are tightly bound to the RTX domain. The function of each Ca(2+) ion is further analyzed using the MD simulation. The removal of Ca3, Ca4, and Ca5 caused the AMS8 lipase structure to become unstable and unfolded. The results suggested that Ca3, Ca4, and Ca5 stabilized the RTX domain. In conclusion, Ca3, Ca4, and Ca5 play a crucial role in the folding and stabilization of the RTX domain, which sustain the integrity of the overall AMS8 lipase structure.
Title: Ion-Pair Interaction and Hydrogen Bonds as Main Features of Protein Thermostability in Mutated T1 Recombinant Lipase Originating from Geobacillus zalihae Ishak SNH, Kamarudin NHA, Ali MSM, Leow ATC, Rahman RNZRA Ref: Molecules, 25:3430, 2020 : PubMed
A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 degC and 76.0 degC in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in beta-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.
Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter 'ragi' from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 degreeC and 70 degreeC) and Hygromycin B (50 g/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhalpha). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhalpha, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of P(FLD1). It could also be promising to express other recombinant proteins without inducers.
Thermostable T1 lipase from Geobacillus zalihae has been crystallized using counter-diffusion method under space and Earth conditions. The comparison of the three-dimensional structures from both crystallized proteins show differences in the formation of hydrogen bond and ion interactions. Hydrogen bond and ion interaction are important in the stabilization of protein structure towards extreme temperature and organic solvents. In this study, the differences of hydrogen bond interactions at position Asp43, Thr118, Glu250, and Asn304 and ion interaction at position Glu226 was chosen to imitate space-grown crystal structure, and the impact of these combined interactions in T1 lipase-mutated structure was studied. Using space-grown T1 lipase structure as a reference, subsequent simultaneous mutation D43E, T118N, E226D, E250L, and N304E was performed on recombinant wild-type T1 lipase (wt-HT1) to generate a quintuple mutant term as 5M mutant lipase. This mutant lipase shared similar characteristics to its wild-type in terms of optimal pH and temperature. The stability of mutant 5M lipase improved significantly in acidic and alkaline pH as compared to wt-HT1. 5M lipase was highly stable in organic solvents such as dimethyl sulfoxide (DMSO), methanol, and n-hexane compared to wt-HT1. Both wild-type and mutant lipases were found highly activated in calcium as compared to other metal ions due to the presence of calcium-binding site for thermostability. The presence of calcium prolonged the half-life of mutant 5M and wt-HT1, and at the same time increased their melting temperature (Tm). The melting temperature of 5M and wt-HT1 lipases increased at 8.4 and 12.1 degrees C, respectively, in the presence of calcium as compared to those without. Calcium enhanced the stability of mutant 5M in 25% (v/v) DMSO, n-hexane, and n-heptane. The lipase activity of wt-HT1 also increased in 25% (v/v) ethanol, methanol, acetonitrile, n-hexane, and n-heptane in the presence of calcium. The current study showed that the accumulation of amino acid substitutions D43E, T118N, E226D, E250L, and N304E produced highly stable T1 mutant when hydrolyzing oil in selected organic solvents such as DMSO, n-hexane, and n-heptane. It is also believed that calcium ion plays important role in regulating lipase thermostability.
Sago starch is traditionally used as food especially in Southeast Asia. Generally, sago is safe for consumption, biodegradable, easily available and inexpensive. Therefore, this research was done to expand the potential of sago by using it as a support for enzyme immobilization. In this study, ARM lipase, which was isolated from Geobacillus sp. strain ARM, was overexpressed in Escherichia coli system and then purified using affinity chromatography. The specific activity of the pure enzyme was 650 U/mg, increased 7 folds from the cell lysate. The purified enzyme was immobilized in gelatinized sago and spray-dried by entrapment technique in order to enhance the enzyme operational stability for handling at high temperature and also for storage. The morphology of the gelatinized sago and immobilized enzyme was studied by scanning electron microscopy. The results showed that the spray-dried gelatinized sago was shrunken and became irregular in structure as compared to untreated sago powder. The surface areas and porosities of spray-dried gelatinized sago with and without the enzyme were analyzed using BET and BJH method and have shown an increase in surface area and decrease in pore size. The immobilized ARM lipase showed good performance at 60-80 degrees C, with a half-life of 4 h and in a pH range 6-9. The immobilized enzyme could be stored at 10 degrees C with the half-life for 9 months. Collectively, the spray-dried immobilized lipase shows promising capability for industrial uses, especially in food processing.
Thermostability remains one of the most desirable traits in many lipases. Numerous studies have revealed promising strategies to improve thermostability and random mutagenesis often leads to unexpected yet interesting findings in engineering stability. Previously, the thermostability of C-terminal truncated cold-adapted lipase from Staphylococcus epidermidis AT2 (rT-M386) was markedly enhanced by directed evolution. The newly evolved mutant, G210C, demonstrated an optimal temperature shift from 25 to 45 degrees C and stability up to 50 degrees C. Interestingly, a cysteine residue was randomly introduced on the loop connecting the two lids and accounted for the only cysteine found in the lipase. We further investigated the structural and mechanistic insights that could possibly cause the significant temperature shift. Both rT-M386 and G210C were modeled and simulated at 25 degrees C and 50 degrees C. The results clearly portrayed the effect of cysteine substitution primarily on the lid stability. Comparative molecular dynamics simulation analysis revealed that G210C exhibited greater stability than the wild-type at high temperature simulation. The compactness of the G210C lipase structure increased at 50 degrees C and resulted in enhanced rigidity hence stability. This observation is supported by the improved and stronger non-covalent interactions formed in the protein structure. Our findings suggest that the introduction of a single cysteine residue at the lid region of cold-adapted lipase may result in unexpected increased in thermostability, thus this approach could serve as one of the thermostabilization strategies in engineering lipase stability.
Title: Effects of Lid 1 Mutagenesis on Lid Displacement, Catalytic Performances and Thermostability of Cold-active Pseudomonas AMS8 Lipase in Toluene Yaacob N, Kamarudin NHA, Leow ATC, Salleh AB, Rahman RNZRA, Ali MSM Ref: Comput Struct Biotechnol J, 17:215, 2019 : PubMed
Pseudomonas fluorescens AMS8 lipase lid 1 structure is rigid and holds unclear roles due to the absence of solvent-interactions. Lid 1 region was stabilized by 17 hydrogen bond linkages and displayed lower mean hydrophobicity (0.596) compared to MIS38 lipase. Mutating lid 1 residues, Thr-52 and Gly-55 to aromatic hydrophobic-polar tyrosine would churned more side-chain interactions between lid 1 and water or toluene. This study revealed that T52Y leads G55Y and its recombinant towards achieving higher solvent-accessible surface area and longer half-life at 25 to 37 degrees C in 0.5% (v/v) toluene. T52Y also exhibited better substrate affinity with long-chain carbon substrate in aqueous media. The affinity for pNP palmitate, laurate and caprylate increased in 0.5% (v/v) toluene in recombinant AMS8, but the affinity in similar substrates was substantially declined in lid 1 mutated lipases. Regarding enzyme efficiency, the recombinant AMS8 lipase displayed highest value of kcat/Km in 0.5% (v/v) toluene, mainly with pNPC. In both hydrolysis reactions with 0% and 0.5% (v/v) toluene, the enzyme efficiency of G55Y was found higher than T52Y for pNPL and pNPP. At 0.5% (v/v) toluene, both mutants showed reductions in activation energy and enthalpy values as temperature increased from 25 to 35 degrees C, displaying better catalytic functions. Only T52Y exhibited increase in entropy values at 0.5% (v/v) toluene indicating structure stability. As a conclusion, Thr-52 and Gly-55 are important residues for lid 1 stability as their existence helps to retain the geometrical structure of alpha-helix and connecting hinge.
GDSL esterase J15 (EstJ15) is a member of Family II of lipolytic enzyme. The enzyme was further classified in subgroup SGNH hydrolase due to the presence of highly conserve motif, Ser-Gly-Asn-His in four conserved blocks I, II, III, and V, respectively. X-ray quality crystal of EstJ15 was obtained from optimized formulation containing 0.10M ammonium sulphate, 0.15M sodium cacodylate trihydrate pH6.5, and 20% PEG 8000. The crystal structure of EstJ15 was solved at 1.38A with one molecule per asymmetric unit. The structure exhibits alpha/beta hydrolase fold and shared low amino acid sequence identity of 23% with the passenger domain of the autotransporter EstA of Pseudomonas aeruginosa. The active site is located at the centre of the structure, formed a narrow tunnel that hinder long substrates to be catalysed which was proven by the protein-ligand docking analysis. This study facilitates the understanding of high substrate specificity of EstJ15 and provide insights on its catalytic mechanism.
Title: The Effects of One Amino Acid Substitutions at the C-Terminal Region of Thermostable L2 Lipase by Computational and Experimental Approach Sani HA, Shariff FM, Rahman RNZRA, Leow ATC, Salleh AB Ref: Mol Biotechnol, 60:1, 2018 : PubMed
The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 degrees C in Tris-HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 degrees C temperature in Tris-HCl pH 8, while for L2 lipase it was at 70 degrees C in Glycine-NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m) value was at 85.96 degrees C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 degrees C within 16 h preincubation.
Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacilluszalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition.
The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD) of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol) and water mixture systems to a concentration of 50%. On the basis of the MD results, the structural deviations of the backbone atoms elucidated the dynamic effects of water/organic solvent mixtures on the equilibrium state of the protein simulations in decreasing solvent polarity. The results show that the solvent mixture gives rise to deviations in enzyme structure from the native one simulated in water. The drop in the flexibility in H2O, MtOH, EtOH and PrOH simulation mixtures shows that greater motions of residues were influenced in BtOH and PtOH simulation mixtures. Comparing the root mean square fluctuations value with the accessible solvent area (SASA) for every residue showed an almost correspondingly high SASA value of residues to high flexibility and low SASA value to low flexibility. The study further revealed that the organic solvents influenced the formation of more hydrogen bonds in MtOH, EtOH and PrOH and thus, it is assumed that increased intraprotein hydrogen bonding is ultimately correlated to the stability of the protein. However, the solvent accessibility analysis showed that in all solvent systems, hydrophobic residues were exposed and polar residues tended to be buried away from the solvent. Distance variation of the tetrahedral intermediate packing of the active pocket was not conserved in organic solvent systems, which could lead to weaknesses in the catalytic H-bond network and most likely a drop in catalytic activity. The conformational variation of the lid domain caused by the solvent molecules influenced its gradual opening. Formation of additional hydrogen bonds and hydrophobic interactions indicates that the contribution of the cooperative network of interactions could retain the stability of the protein in some solvent systems. Time-correlated atomic motions were used to characterize the correlations between the motions of the atoms from atomic coordinates. The resulting cross-correlation map revealed that the organic solvent mixtures performed functional, concerted, correlated motions in regions of residues of the lid domain to other residues. These observations suggest that varying lengths of polar organic solvents play a significant role in introducing dynamic conformational diversity in proteins in a decreasing order of polarity.
Title: Impact of signal peptide and transmembrane segments on expression and biochemical properties of a lipase from Bacillus sphaericus 205y Masomian M, Jasni AS, Rahman RNZRA, Salleh AB, Basri M Ref: J Biotechnol, 264:51, 2017 : PubMed
A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an alpha/beta hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein.
Title: Improving the Efficiency of New Automatic Dishwashing Detergent Formulation by Addition of Thermostable Lipase, Protease and Amylase Naganthran A, Masomian M, Rahman RNZRA, Ali MSM, Nooh HM Ref: Molecules, 22:, 2017 : PubMed
The use of T1 lipase in automatic dishwashing detergent (ADD) is well established, but efficiency in hard water is very low. A new enzymatic environmentally-friendly dishwashing was formulated to be efficient in both soft and hard water. Thermostable enzymes such as T1 lipase from Geobacillus strain T1, Rand protease from Bacillussubtilis strain Rand, and Maltogenic amylase from Geobacillus sp. SK70 were produced and evaluated for an automatic dishwashing detergent formulation. The components of the new ADD were optimized for compatibility with these three enzymes. In compatibility tests of the enzymes with different components, several criteria were considered. The enzymes were mostly stable in non-ionic surfactants, especially polyhydric alcohols, Glucopon UP 600, and in a mixture of sodium carbonate and glycine (30:70) buffer at a pH of 9.25. Sodium polyacrylate and sodium citrate were used in the ADD formulation as a dispersing agent and a builder, respectively. Dishwashing performance of the formulated ADDs was evaluated in terms of percent of soil removed using the Leenert's Improved Detergency Tester. The results showed that the combination of different hydrolysis enzymes could improve the washing efficiency of formulated ADD compared to the commercial ADD "Finish" at 40 and 50 C.
Title: The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene Yaacob N, Kamarudin NHA, Leow ATC, Salleh AB, Rahman RNZRA, Ali MSM Ref: Molecules, 22:, 2017 : PubMed
The alkaline cold-active lipase from Pseudomonas fluorescens AMS8 undergoes major structural changes when reacted with hydrophobic organic solvents. In toluene, the AMS8 lipase catalytic region is exposed by the moving hydrophobic lid 2 (Glu-148 to Gly-167). Solvent-accessible surface area analysis revealed that Leu-208, which is located next to the nucleophilic Ser-207 has a focal function in influencing substrate accessibility and flexibility of the catalytic pocket. Based on molecular dynamic simulations, it was found that Leu-208 strongly facilitates the lid 2 opening via its side-chain. The KM and Kcat/KM of L208A mutant were substrate dependent as it preferred a smaller-chain ester (pNP-caprylate) as compared to medium (pNP-laurate) or long-chain (pNP-palmitate) esters. In esterification of ethyl hexanoate, L208A promotes a higher ester conversion rate at 20 degrees C but not at 30 degrees C, as a 27% decline was observed. Interestingly, the wild-type (WT) lipase's conversion rate was found to increase with a higher temperature. WT lipase AMS8 esterification was higher in toluene as compared to L208A. Hence, the results showed that Leu-208 of AMS8 lipase plays an important role in steering a broad range of substrates into its active site region by regulating the flexibility of this region. Leu-208 is therefore predicted to be crucial for its role in interfacial activation and catalysis in toluene.
Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30 degreesC at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40 degreesC, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li(+), Na(+), K(+), Rb(+) and Cs(+) after 30min treatment. Heavy metal ions such as Cu(2+), Fe(3+) and Zn(2+) inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature.
Title: Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases Masomian M, Rahman RNZRA, Salleh AB, Basri M Ref: PLoS ONE, 11:e0149851, 2016 : PubMed
Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with <= 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca2+-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an alpha/beta hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 degrees C and retained >/= 97% activity after incubation at 50 degrees C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.
Title: Facile modulation of enantioselectivity of thermophilic Geobacillus zalihae lipase by regulating hydrophobicity of its Q114 oxyanion Wahab RA, Basri M, Rahman RNZRA, Salleh AB, Abdul Rahman MB, Leow ATC Ref: Enzyme Microb Technol, 93-94:174, 2016 : PubMed
Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50 degrees C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.
Title: Toluene promotes lid 2 interfacial activation of cold active solvent tolerant lipase from Pseudomonas fluorescens strain AMS8 Yaacob N, Ali MSM, Salleh AB, Rahman RNZRA, Leow AT Ref: J Mol Graph Model, 68:224, 2016 : PubMed
The utilization of cold active lipases in organic solvents proves an excellent approach for chiral synthesis and modification of fats and oil due to the inherent flexibility of lipases under low water conditions. In order to verify whether this lipase can function as a valuable synthetic catalyst, the mechanism concerning activation of the lid and interacting solvent residues in the presence of organic solvent must be well understood. A new alkaline cold-adapted lipase, AMS8, from Pseudomonas fluorescens was studied for its structural adaptation and flexibility prior to its exposure to non-polar, polar aprotic and protic solvents. Solvents such as ethanol, toluene, DMSO and 2-propanol showed to have good interactions with active sites. Asparagine (Asn) and tyrosine (Tyr) were key residues attracted to solvents because they could form hydrogen bonds. Unlike in other solvents, Phe-18, Tyr-236 and Tyr-318 were predicted to have aromatic-aromatic side-chain interactions with toluene. Non-polar solvent also was found to possess highest energy binding compared to polar solvents. Due to this circumstance, the interaction of toluene and AMS8 lipase was primarily based on hydrophobicity and molecular recognition. The molecular dynamic simulation showed that lid 2 (residues 148-167) was very flexible in toluene and Ca2+. As a result, lid 2 moves away from the catalytic areas, leaving an opening for better substrate accessibility which promotes protein activation. Only a single lid (lid 2) showed the movement following interactions with toluene, although AMS8 lipase displayed double lids. The secondary conformation of AMS8 lipase that was affected by toluene observed a reduction of helical strands and increased coil structure. Overall, this work shows that cold active lipase, AMS8 exhibits distinguish interfacial activation and stability in the presence of polar and non-polar solvents.
Psychrophilic microorganisms are cold-adapted with distinct properties from other thermal classes thriving in cold conditions in large areas of the earth's cold environment. Maintenance of functional membranes, evolving cold-adapted enzymes and synthesizing a range of structural features are basic adaptive strategies of psychrophiles. Among the cold-evolved enzymes are the cold-active lipases, a group of microbial lipases with inherent stability-activity-flexibility property that have engaged the interest of researchers over the years. Current knowledge regarding these cold-evolved enzymes in psychrophilic bacteria proves a display of high catalytic efficiency with low thermal stability, which is a differentiating feature with that of their mesophilic and thermophilic counterparts. Improvement strategies of their adaptive structural features have significantly benefited the enzyme industry. Based on their homogeneity and purity, molecular characterizations of these enzymes have been successful and their properties make them unique biocatalysts for various industrial and biotechnological applications. Although, strong association of lipopolysaccharides from Antarctic microorganisms with lipid hydrolases pose a challenge in their purification, heterologous expression of the cold-adapted lipases with affinity tags simplifies purification with higher yield. The review discusses these cold-evolved lipases from bacteria and their peculiar properties, in addition to their potential biotechnological and industrial applications.
Geobacillus zalihae sp. nov., which produces a putative thermostable lipase, represents a novel species, with type strain T1. The characterisation of this intrinsically thermostable T1 lipase either physicochemically or structurally is an important task. The crystallisation of T1lipase in space was carried out using a High-Density Protein Crystal Growth (HDPCG) apparatus with the vapour diffusion method, and X-ray diffraction data were collected. The microgravity environment has improved the size and quality of the crystals as compared to earth grown crystal. The effect of microgravity on the crystallisation of T1 lipase was clearly evidenced by the finer atomic details at 1.35 A resolution. Better electron densities were observed overall compared with the Earth-grown crystals, and comparison shows the subtle but distinct conformations around Na(+) ion binding site stabilized via cation-pi interactions. This approach could be useful for solving structure and function of lipases towards exploiting its potentials to various industrial applications.
Three-dimensional structure of thermostable lipase is much sought after nowadays as it is important for industrial application mainly found in the food, detergent, and pharmaceutical sectors. Crystallization utilizing the counter diffusion method in space was performed with the aim to obtain high resolution diffracting crystals with better internal order to improve the accuracy of the structure. Thermostable T1 lipase enzyme has been crystallized in laboratory on earth and also under microgravity condition aboard Progress spacecraft to the ISS in collaboration with JAXA (Japanese Aerospace Exploration Agency). This study is conducted with the aims of improving crystal packing and structure resolution. The diffraction data set for ground grown crystal was collected to 1.3 A resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.40 A, b = 80.95 A, and c = 99.81 A, whereas the diffraction data set for space grown crystal was collected to 1.1 A resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.31 A, b = 80.85 A, and c = 99.81 A. The major difference between the two crystal growth systems is the lack of convection and sedimentation in microgravity environment resulted in the growth of much higher quality crystals of T1 lipase.
Title: Enzymatic production of a solvent-free menthyl butyrate via response surface methodology catalyzed by a novel thermostable lipase from Geobacillus zalihae Wahab RA, Basri M, Rahman RNZRA, Salleh AB, Rahman MB, Chaibakhsh N, Leow ATC Ref: Biotechnol Biotechnol Equip, 28:1065, 2014 : PubMed
Most substrate for esterification has the inherent problem of low miscibility which requires addition of solvents into the reaction media. In this contribution, we would like to present an alternative and feasible option for an efficient solvent-free synthesis of menthyl butyrate using a novel thermostable crude T1 lipase. We investigated the effects of incubation time, temperature, enzyme loading and substrate molar ratio and determined the optimum conditions. The high conversion of menthyl butyrate catalyzed by crude T1 lipase in a solvent-free system is greatly affected by temperature and time of the reaction media. The highest yield of menthyl butyrate was 99.3% under optimized conditions of 60 degreesC, incubation time of 13.15 h, 2.53 mg, 0.43% (w/w) enzyme to substrate ratio and at molar ratio of butyric anhydride/menthol 2.7:1. Hence, the investigation revealed that the thermostable crude T1 lipase successfully catalyzed the high-yield production of menthyl butyrate in a solvent-free system. The finding suggests that the crude T1 lipase was a promising alternative to overcome shortcomings associated with solvent-assisted enzymatic reactions.
Title: Structural Adaptation of Cold-Active RTX Lipase from Pseudomonas sp. Strain AMS8 Revealed via Homology and Molecular Dynamics Simulation Approaches Ali MSM, Mohd Fuzi SF, Ganasen M, Rahman RNZRA, Basri M, Salleh AB Ref: Biomed Res Int, 2013:925373, 2013 : PubMed
The psychrophilic enzyme is an interesting subject to study due to its special ability to adapt to extreme temperatures, unlike typical enzymes. Utilizing computer-aided software, the predicted structure and function of the enzyme lipase AMS8 (LipAMS8) (isolated from the psychrophilic Pseudomonas sp., obtained from the Antarctic soil) are studied. The enzyme shows significant sequence similarities with lipases from Pseudomonas sp. MIS38 and Serratia marcescens. These similarities aid in the prediction of the 3D molecular structure of the enzyme. In this study, 12 ns MD simulation is performed at different temperatures for structural flexibility and stability analysis. The results show that the enzyme is most stable at 0 degrees C and 5 degrees C. In terms of stability and flexibility, the catalytic domain (N-terminus) maintained its stability more than the noncatalytic domain (C-terminus), but the non-catalytic domain showed higher flexibility than the catalytic domain. The analysis of the structure and function of LipAMS8 provides new insights into the structural adaptation of this protein at low temperatures. The information obtained could be a useful tool for low temperature industrial applications and molecular engineering purposes, in the near future.
Title: A new thermostable and organic solvent-tolerant lipase from Aneurinibacillus thermoaerophilus strain HZ. Masomian M, Rahman RNZRA, Salleh AB, Basri M Ref: Process Biochemistry, 48:169, 2013 : PubMed
A thermostable and organic solvent-tolerant lipase produced by Aneurinibacillus thermoaerophilus strain HZ was purified and characterised. The lipase was purified to apparent homogeneity with two steps: anion exchange chromatography on Q-Sepharose and gel filtration on Sephadex-G75. A final specific activity of 43.5 U/mg was obtained with an overall recovery of 19.7% and 15.6 purification fold. The molecular mass of the HZ lipase was estimated to be 50 kDa. The optimum pH for the activity of the purified HZ lipase was 7.0. The stability showed a broad range of pH values between pH 4.0 and 9.0 at 30 C. The purified HZ lipase exhibited an optimum temperature of 65 C with a half-life of 3 h and 10 min at 65 C. The activity of the purified HZ lipase was stimulated in the presence of Ca2+. Organic solvents such as dimethyl sulfoxide (DMSO), methanol, n-tetradecane and n-hexadecane enhanced the lipase activity. Studies on the effect of oil showed that the lipase preferred natural oil, such as sunflower oil, over synthetic substrates.
The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100 degrees C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises alpha6 and alpha7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.
A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 degrees C and showed stability over a temperature range of 40-60 degrees C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
Title: 3D structure elucidation of thermostable L2 lipase from Thermophilic Bacillus sp. L2 Rahman RNZRA, Shariff FM, Basri M, Salleh AB Ref: Int J Mol Sci, 13:9207, 2012 : PubMed
The crystallization of proteins makes it possible to determine their structure by X-ray crystallography, and is therefore important for the analysis of protein structure-function relationships. L2 lipase was crystallized by using the J-tube counter diffusion method. A crystallization consisting of 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl was found to be the best condition to produce crystals with good shape and size (0.5 x 0.1 x 0.2 mm). The protein concentration used for the crystallization was 3 mg/mL. L2 lipase crystal has two crystal forms, Shape 1 and Shape 2. Shape 2 L2 lipase crystal was diffracted at 1.5 A and the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 72.0, b = 81.8, c = 83.4 A, alpha = beta = gamma = 90 degrees . There is one molecule per asymmetric unit and the solvent content of the crystals is 56.9%, with a Matthew's coefficient of 2.85 A Da(-1). The 3D structure of L2 lipase revealed topological organization of alpha/beta-hydrolase fold consisting of 11 beta-strands and 13 alpha-helices. Ser-113, His-358 and Asp-317 were assigned as catalytic triad residues. One Ca(2+) and one Zn(2+) were found in the L2 lipase molecule.
Title: Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 Ruslan R, Rahman RNZRA, Leow ATC, Ali MSM, Basri M, Salleh AB Ref: Int J Mol Sci, 13:943, 2012 : PubMed
Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 degrees C, 70.59 degrees C and 68.54 degrees C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 A using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 A, b = 81.16 A and c = 100.14 A. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.
Title: Combination of Oxyanion Gln114 Mutation and Medium Engineering to Influence the Enantioselectivity of Thermophilic Lipase from Geobacillus zalihae Wahab RA, Basri M, Rahman MB, Rahman RNZRA, Salleh AB, Leow ATC Ref: Int J Mol Sci, 13:11666, 2012 : PubMed
The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.The substitution of the oxyanion Q114 wit
Title: Manipulation of the conformation and enzymatic properties of T1 lipase by site-directed mutagenesis of the protein core Wahab RA, Basri M, Rahman RNZRA, Salleh AB, Rahman MB, Chor LT Ref: Appl Biochem Biotechnol, 167:612, 2012 : PubMed
In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.
Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 degrees C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications.
Immobilized Candida antarctica lipase B-catalyzed esterification of xylitol and two fatty acids (capric and caproic acid) were studied in a solvent-free system. The Taguchi orthogonal array method based on three-level-four-variables with nine experiments was applied for the analysis and optimization of the reaction parameters including time, substrate molar ratio, amount of enzyme, and amount of molecular sieve. The obtained conversion was higher in the esterification of xylitol and capric acid with longer chain length. The optimum conditions derived via the Taguchi approach for the synthesis of xylitol caprate and xylitol caproate were reaction time, 29 and 18h; substrate molar ratio, 0.3 and 1.0; enzyme amount, 0.20 and 0.05g, and molecular sieve amount of 0.03g, respectively. The good correlation between the predicted conversions (74.18% and 61.23%) and the actual values (74.05% and 60.5%) shows that the model derived from the Taguchi orthogonal array can be used for optimization and better understanding of the effect of reaction parameters on the enzymatic synthesis of xylitol esters in a solvent-free system.
Title: Reductive alkylation causes the formation of a molten globule-like intermediate structure in Geobacillus zalihae strain T1 thermostable lipase Cheong KW, Leow ATC, Rahman RNZRA, Basri M, Rahman MB, Salleh AB Ref: Appl Biochem Biotechnol, 164:362, 2011 : PubMed
A thermostable lipase from Geobacillus zalihae strain T1 was chemically modified using propionaldehyde via reductive alkylation. The targeted alkylation sites were lysines, in which T1 lipase possessed 11 residues. Far-UV circular dichroism (CD) spectra of both native and alkylated enzyme showed a similar broad minimum between 208 and 222 nm, thus suggesting a substantial amount of secondary structures in modified enzyme, as compared with the corresponding native enzyme. The hydrolytic activity of the modified enzymes dropped drastically by nearly 15-fold upon chemical modification, despite both the native and modified form showed distinctive alpha-helical bands at 208 and 222 nm in CD spectra, leading us to the hypothesis of formation of a molten globule (MG)-like structure. As cooperative unfolding transitions were observed, the modified lipase was distinguished from the native state, in which the former possessed a denaturation temperature (T(m)) in lower temperature range at 61 degrees C while the latter at 68 degrees C. This was further supported by 8-anilino-1-naphthalenesulfonic acid (ANS) probed fluorescence which indicated higher exposure of hydrophobic residues, consequential of chemical modification. Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a small number of lysine residues were confirmed to be alkylated.
Title: High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM Ebrahimpour A, Rahman RNZRA, Basri M, Salleh AB Ref: Bioresour Technol, 102:6972, 2011 : PubMed
The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65 degrees C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50 degrees C for more than 150 min.
Title: Crystallization and preliminary X-ray crystallographic analysis of a thermostable organic solvent-tolerant lipase from Bacillus sp. strain 42 Khusaini MS, Rahman RNZRA, Ali MSM, Leow ATC, Basri M, Salleh AB Ref: Acta Crystallographica Sect F Struct Biol Cryst Commun, 67:401, 2011 : PubMed
An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 A resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09x0.19x0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=117.41, b=80.85, c=99.44 A, beta=96.40 degrees .
A thermophilic lipolytic bacterium identified as Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5-99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55-80 degrees C and at a pH of 6-10. The L2 lipase had a melting temperature (Tm) of 59.04 degrees C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 degrees C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% alpha-helices, 2.2% ss-strands, 23.6% turns and 35.6% random conformations.
Title: Molten globule-triggered inactivation of a thermostable and solvent stable lipase in hydrophilic solvents Hamid TH, Rahman RNZRA, Salleh AB, Basri M Ref: Protein J, 29:290, 2010 : PubMed
The use of lipase in hydrophilic solvent is usually hampered by inactivation. The solvent stability of a recombinant solvent stable lipase isolated from thermostable Bacillus sp. strain 42 (Lip 42), in DMSO and methanol were studied at different solvent-water compositions. The enzymatic activities were retained in up to 45% v/v solvent compositions. The near-UV CD spectra indicated that tertiary structures were perturbed at 60% v/v and above. Far-UV CD in methanol indicated the secondary structure in Lip 42 was retained throughout all solvent compositions. Fluorescence studies indicated formations of molten globules in solvent compositions of 60% v/v and above. The enzyme was able to retain its secondary structures in the presence of methanol; however, there was a general reduction in beta-sheet and an increase in alpha-helix contents. The H-bonding arrangements triggered in methanol and DMSO, respectively, caused different forms of tertiary structure perturbations on Lip 42, despite both showing partial denaturation with molten globule formations.
An all-atom level MD simulation in explicit solvent at high temperature is a powerful technique to increase our knowledge about the structurally important regions modulating thermal stability in thermenzymes. In this respect, two large-sized thermoalkalophilic enzymes from Bacillus stearothermophilus L1 (L1 lipase) and Geobacillus zalihae strain T1 (T1 lipase) are well-established representatives. In this paper, comparative results from temperature-induced MD simulations of both model systems at 300 K, 400 K and 500 K are presented and discussed with respect to identification of highly flexible regions critical to thermostability. From our MD simulation results, specific regions along the L1 lipase and T1 lipase polypeptide chain including the small domain and the main catalytic domain or core domain of both enzymes show a marked increase in fluctuations and dynamics followed by clear structural changes. Overall, the N-terminal moiety of both enzymes and their small domains exhibit hyper-sensitivity to thermal stress. The results appear to propose that these regions are critical in determining of the overall thermal stability of both organisms.
An organic solvent tolerant lipase gene from Staphylococcus epidermidis AT2 was successfully cloned and expressed with pTrcHis2 in E. coli TOP10. Sequence analysis revealed an open reading frame (ORF) of 1,933 bp in length which coded for a polypeptide of 643 amino acid residues. The polypeptide comprised of a signal peptide (37 amino acids), pro-peptide and a mature protein of 390 amino acids. Expression of AT2 lipase resulted in an 18-fold increase in activity, upon the induction of 0.6 mM IPTG after a 10 h incubation period. Interestingly, this lipase was stable in various organic solvents (25% (v/v), mainly toluene, octanol, p-xylene and n-hexane). Literature shows that most of the organic solvent stable bacterial lipases were produced by Pseudomonas sp. and Bacillus sp., but very few from Staphylococcus sp. This lipase demonstrates great potential to be employed in various industrial applications.
Title: Crystallization and preliminary X-ray crystallographic analysis of highly thermostable L2 lipase from the newly isolated Bacillus sp. L2 Shariff FM, Rahman RNZRA, Ali MSM, Chor AL, Basri M, Salleh AB Ref: Acta Crystallographica Sect F Struct Biol Cryst Commun, 66:715, 2010 : PubMed
Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 A resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 A. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 57%.
In this study, an artificial neural network (ANN) trained by backpropagation algorithm, Levenberg-Marquadart, was applied to predict the yield of enzymatic synthesis of dioctyl adipate. Immobilized Candida antarctica lipase B was used as a biocatalyst for the reaction. Temperature, time, amount of enzyme, and substrate molar ratio were the four input variables. After evaluating various ANN configurations, the best network was composed of seven hidden nodes using a hyperbolic tangent sigmoid transfer function. The correlation coefficient (R2) and mean absolute error (MAE) values between the actual and predicted responses were determined as 0.9998 and 0.0966 for training set and 0.9241 and 1.9439 for validating dataset. A simulation test with a testing dataset showed that the MAE was low and R2 was close to 1. These results imply the good generalization of the developed model and its capability to predict the reaction yield. Comparison of the performance of radial basis network with the developed models showed that radial basis function was more accurate but its performance was poor when tested with unseen data. In further part of the study, the feedforward backpropagation model was used for prediction of the ester yield within the given range of the main parameters.
Immobilized Candida antarctica lipase-catalyzed esterification of adipic acid and oleyl alcohol was investigated in a solvent-free system (SFS). Optimum conditions for adipate ester synthesis in a stirred-tank reactor were determined by the response surface methodology (RSM) approach with respect to important reaction parameters including time, temperature, agitation speed, and amount of enzyme. A high conversion yield was achieved using low enzyme amounts of 2.5% w/w at 60 degrees C, reaction time of 438 min, and agitation speed of 500 rpm. The good correlation between predicted value (96.0%) and actual value (95.5%) implies that the model derived from RSM allows better understanding of the effect of important reaction parameters on the lipase-catalyzed synthesis of adipate ester in an organic solvent-free system. Higher volumetric productivity compared to a solvent-based system was also offered by SFS. The results demonstrate that the solvent-free system is efficient for enzymatic synthesis of adipate ester.
Title: Characterization and solvent stable features of Strep-tagged purified recombinant lipase from thermostable and solvent tolerant Bacillus sp. strain 42. Hamid THTA, Eltaweel MA, Rahman RNZRA, Basri M, Salleh AB Ref: Ann Microbiol, 59:111, 2009 : PubMed
A 1.2 kb lipase gene (AY 78735) from solvent stable and thermostableBacillus sp. strain 42 was overexpressed in a heterologous system that allowed for an extensive characterization of its solvent stability and thermostability. An overexpression was achieved using pET51b vector withEscherichia coli host strain BL21(DE3)pLysS, in which optimum expression was at 22-24 h incubation at 37C, with lipase activity reached at 80.0 U mL-1 (specific activity 160.0 U mg-1), after induction by 0.5 mM IPTG. This expression was 11.5 fold higher and superseded the pQE-30UA/M15 (pREP4) host-vector system, which only achieved at 17.0 U mL1 (34.0 U mg-1). The fusion lipase contains N-terminal Strep-tag II affinity tag that in one step of purification, the lipase was purified to homogeneity using Strep-tag II agarose column. The lipase was purified at 1.3 fold and 70% recovery with the elution fraction gave a band of 43 kDa in SDS-PAGE. The purified fusion lipase was most active at 70C and pH 8.0, and was stable in a broad pH range of 7-10. It showed hydrolysis preference towards olive, sunflower and corn oils. Based on solvent stability studies in 30 min pre-incubation in 25% v/v solvents with a shaking rate at 150 strokes per min, the purified Lip 42 showed a different residual activity profiles depending on solvents and temperatures. Lip 42 was found be stable in polar organic solvents such as DMSO, DMF, acetone, methanol, heptanol and octanol, which could make it as a potential biocatalyst for the use in industrial biodiesel production.
Title: Secretory expression and characterization of a highly Ca2+-activated thermostable L2 lipase Sabri S, Rahman RNZRA, Leow ATC, Basri M, Salleh AB Ref: Protein Expr Purif, 68:161, 2009 : PubMed
Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae alpha-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 degrees C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca2+. L2 lipase showed a preference for medium to long chain triacylglycerols (C(10)-C(16)), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.
Title: A modeling study by response surface methodology and artificial neural network on culture parameters optimization for thermostable lipase production from a newly isolated thermophilic Geobacillus sp. strain ARM Ebrahimpour A, Rahman RNZRA, Ean Ch'ng DH, Basri M, Salleh AB Ref: BMC Biotechnol, 8:96, 2008 : PubMed
BACKGROUND: Thermostable bacterial lipases occupy a place of prominence among biocatalysts owing to their novel, multifold applications and resistance to high temperature and other operational conditions. The capability of lipases to catalyze a variety of novel reactions in both aqueous and nonaqueous media presents a fascinating field for research, creating interest to isolate novel lipase producers and optimize lipase production. The most important stages in a biological process are modeling and optimization to improve a system and increase the efficiency of the process without increasing the cost. RESULTS: Different production media were tested for lipase production by a newly isolated thermophilic Geobacillus sp. strain ARM (DSM 21496 = NCIMB 41583). The maximum production was obtained in the presence of peptone and yeast extract as organic nitrogen sources, olive oil as carbon source and lipase production inducer, sodium and calcium as metal ions, and gum arabic as emulsifier and lipase production inducer. The best models for optimization of culture parameters were achieved by multilayer full feedforward incremental back propagation network and modified response surface model using backward elimination, where the optimum condition was: growth temperature (52.3 degrees C), medium volume (50 ml), inoculum size (1%), agitation rate (static condition), incubation period (24 h) and initial pH (5.8). The experimental lipase activity was 0.47 Uml(-1) at optimum condition (4.7-fold increase), which compared well to the maximum predicted values by ANN (0.47 Uml(-1)) and RSM (0.476 Uml(-1)), whereas R2 and AAD were determined as 0.989 and 0.059% for ANN, and 0.95 and 0.078% for RSM respectively. CONCLUSION: Lipase production is the result of a synergistic combination of effective parameters interactions. These parameters are in equilibrium and the change of one parameter can be compensated by changes of other parameters to give the same results. Though both RSM and ANN models provided good quality predictions in this study, yet the ANN showed a clear superiority over RSM for both data fitting and estimation capabilities. On the other hand, ANN has the disadvantage of requiring large amounts of training data in comparison with RSM. This problem was solved by using statistical experimental design, to reduce the number of experiments.
Lipase-catalyzed production of palm esters by alcoholysis of palm oil with oleyl alcohol in n-hexane was performed in 2L stirred-tank reactor (STR). Investigation on the performance of reactor operation was carried out in batch mode STR with single impeller mounted on the centrally located shaft. Rushton turbine (RT) impellers provide the highest reaction yield (95.8%) at lower agitation speed as compared to AL-hydrofoil (AL-H) and 2-bladed elephant ear (EE) impellers. Homogenous enzyme particles suspension was obtained at 250 rpm by using RT impeller. At higher impeller speed, the shear effect on the enzyme particles caused by agitation has decreased the reaction performance. Palm esters reaction mixture in STR follows Newtons' law due to the linear relation between the shear stress (tau) and shear rate (dupsilon/dy). High stability of Lipozyme RM IM was observed as shown by its ability to be repeatedly used to give high percentage yield (79%) of palm esters even after 15 cycles of reaction. The process was successfully scale-up to 75 L STR (50 L working volume) based on a constant impeller tip speed approach, which gave the yield of 97.2% after 5h reaction time.
A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70 degrees C and pH 9, respectively. It was stable up to 65 degrees C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na(+), Ca(2+), Mn(2+), K(+) and Mg(2+ ), but inhibited by Cu(2+), Fe(3+) and Zn(2+). Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T(m) for T1 lipase was around 72.2 degrees C, as revealed by denatured protein analysis of CD spectra.
The gene encoding thermostable T1 lipase secreted by Geobacillus sp. strain T1 has been overexpressed in a prokaryotic system. Preliminary crystallization was conducted with crystal screen and crystal screen II through a sitting drop vapor diffusion method with 0.5 mg/mL purified T1 lipase at 16 C. Crystallization at 16 degC using formulation 21 of crystal screen II at 2.5 mg/mL yielded bigger and more defined crystals. Good crystals could easily be obtained as the temperature was increased further while retaining other conditions. In fact, crystallization of T1 lipase is still possible at 60 C. The ability to form crystals at 60 degC is a new discovery in lipase crystallization.
Title: Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia Rahman RNZRA, Leow ATC, Salleh AB, Basri M Ref: BMC Microbiol, 7:77, 2007 : PubMed
BACKGROUND: Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78 degrees C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5-99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification. RESULTS: Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1T was chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70 degrees C and was also stable up to 60 degrees C without significant loss of crude enzyme activity. Strain T1T was a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1T was shown to belong to the Bacillus rRNA group 5 related to Geobacillus thermoleovorans (DSM 5366T) and Geobacillus kaustophilus (DSM 7263T). Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1T to the genus Geobacillus. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1T from its validly published closest phylogenetic neighbors. Strain T1T therefore represents a novel species, for which the name Geobacillus zalihae sp. nov. is proposed, with the type strain T1T (=DSM 18318T; NBRC 101842T). CONCLUSION: Strain T1T was able to secrete extracellular thermostable lipase into culture medium. The strain T1T was identified as Geobacillus zalihae T1T as it differs from its type strains Geobacillus kaustophilus (DSM 7263T) and Geobacillus thermoleovorans (DSM 5366T) on some physiological studies, cellular fatty acids composition, RiboPrint analysis, length of lipase gene and protein profile.
A thermophilic bacterium, Bacillus sp. strain L2 was isolated from a hot spring in Perak, Malaysia. An extracellular lipase activity was detected through plate and broth assays at 70 degrees C after 28 h of incubation. The L2 lipase production was growth dependent as revealed by a number of factors affecting the secretion of extracelullar lipase. As for nutritional factors, casamino acids, trehalose, Ca(2+) and Tween 60 were found to be more effective for lipase production. The optimum physical condition for L2 lipase production was obtained at 70 degrees C after 28 h of cultivation time, at pH 7.0, 150 rpm of agitation rate and 1% of starting inoculum size. The activity staining of crude L2 lipase revealed a clearing zone at 39 kDa.
In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.
Title: A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene Sulong MR, Rahman RNZRA, Salleh AB, Basri M Ref: Protein Expr Purif, 49:190, 2006 : PubMed
UNLABELLED: An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity. KEYWORDS: Bacter
Title: High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5 Rahman RNZRA, Baharum SN, Basri M, Salleh AB Ref: Analytical Biochemistry, 341:267, 2005 : PubMed
An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 degrees C and 9.0, respectively. The purified lipase was stable at 45 degrees C and pH 6-9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca2+ and Mg2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography.
Title: High level expression of thermostable lipase from Geobacillus sp. strain T1 Leow ATC, Rahman RNZRA, Basri M, Salleh AB Ref: Biosci Biotechnol Biochem, 68:96, 2004 : PubMed
A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg(-1) which corresponds to 2927.15 Ug(-1) of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65 degrees C and pH 9, respectively. It was stable up to 65 degrees C at pH 7 and active over a wide pH range (pH 6-11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.
Title: Cloning and expression of a novel lipase gene from Bacillus sphaericus 205y Rahman RNZRA, Chin JH, Salleh AB, Basri M Ref: Mol Genet Genomics, 269:252, 2003 : PubMed
A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene.
Title: Optimization of the enzyme-catalyzed synthesis of amino acid-based surfactants from palm oil fractions Soo EL, Salleh AB, Basri M, Rahman RNZRA, Kamaruddin K Ref: J Biosci Bioeng, 95:361, 2003 : PubMed
The feasibility of using palm oil fractions as cheap and abundant sources of raw material for the synthesis of amino acid surfactants was investigated. Of a number of enzymes screened, the best results were obtained with the immobilized enzyme, Lipozyme. The effects of temperature, solvent, incubation period, fatty substrate/amino acid molar ratio, enzyme amount, and water removal on the reactions were analyzed and compared to those on reactions with free fatty acids and pure triglycerides as fatty substrates. All reactions were most efficient when carried out at high temperatures (70-80 degrees C) in hexane as a solvent. However, while reactions with free fatty acids proceeded better when a slight excess of the free fatty acids over the amino acids was used, reactions with triglycerides and palm oil fractions were best performed at equimolar ratios. Also, the addition of molecular sieves slightly enhanced reactions with free fatty acids but adversely affected reactions with triglycerides and palm oil fractions. Although reactions with palm oil fractions took longer (6 d) to reach equilibrium compared to reactions with free fatty acids (4 d) and pure triglycerides (4 d), better yields were obtained. Such lipase-catalyzed transacylation of palm oil fractions with amino acids is potentially useful in the production of mixed medium- to long-chain surfactants for specific applications.
