Recent investigations on cloned bacterial lipases performed in our laboratory revealed the presence of lipolytic activity that was not due to the cloned lipase-coding gene but was probably the result of an intrinsic activity of Escherichia coli itself. To confirm such a hypothesis, we assayed the activity of frequently used E. coli strains by fast paper tests, zymograms and spectrofluorometry. A band of Ca. 18-20kDa showing activity on MUF-butyrate was detected in zymogram analysis of crude cell extracts in all E. coli strains assayed. Moreover, the spectrofluorometric results obtained confirmed the presence of low but significant lipolytic activity in E. coli, with strain BL21 showing the highest activity. Detailed characterization of such a lipolytic activity was performed using E. coli BL21 cell extracts, where preference for C7 substrates was found, although shorter substrates were also hydrolysed to a minor extent. Interestingly, E. coli lipolytic activity displays traits of a thermophilic enzyme, showing maximum activity at 50degC and pH 8, an unexpected feature never described before. Kinetic and inhibition analysis were also performed showing that activity can be inhibited by several metal ions or by Triton X-100 and SDS, used in zymogram analysis. Such properties low activity, preference for medium chain-length substrates, and high operational temperature might justify why this activity had gone unexplored until now, even when many lipases and esterases have been cloned and expressed in E. coli strains in the past. From now on, lipase researchers should take into consideration the presence of such a basal lipolytic activity before starting their lipase cloning or expression experiments in E.coli.
Title: Rational evolution of the unusual Y-type oxyanion hole of Rhodococcus sp. CR53 lipase LipR Infanzon B, Sotelo PH, Martinez J, Diaz P Ref: Enzyme Microb Technol, 108:26, 2018 : PubMed
Rhodococcus sp CR-53 lipase LipR was the first characterized member of bacterial lipase family X. Interestingly, LipR displays some similarity with alpha/beta-hydrolases of the C. antartica lipase A (CAL-A)-like superfamily (abH38), bearing a Y-type oxyanion hole, never found before among bacterial lipases. In order to explore this unusual Y-type oxyanion hole, and to improve LipR performance, two modification strategies based on site directed or saturation mutagenesis were addressed. Initially, a small library of mutants was designed to convert LipR Y-type oxyanion hole (YDS) into one closer to those most frequently found in bacteria (GGG(X)). However, activity was completely lost in all mutants obtained, indicating that the Y-type oxyanion hole of LipR is required for activity. A second approach was addressed to modify the two main oxyanion hole residues Tyr110 and Asp111, previously described for CAL-A as the most relevant amino acids involved in stabilization of the enzyme-substrate complex. A saturation mutagenesis library was prepared for each residue (Tyr110 and Asp111), and activity of the resulting variants was assayed on different chain length substrates. No functional LipR variants could be obtained when Tyr110 was replaced by any other amino acids, indicating that this is a crucial residue for catalysis. However, among the Asp111 variants obtained, LipR D111G produced a functional enzyme. Interestingly, this LipR-YGS variant showed less activity than wild type LipR on short- or mid- chain substrates but displayed a 5.6-fold increased activity on long chain length substrates. Analysis of the 3D model and in silico docking studies of this enzyme variant suggest that substitution of Asp by Gly produces a wider entrance tunnel that would allow for a better and tight accommodation of larger substrates, thus justifying the experimental results obtained.
Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80 degrees C and pH 8-9, being strongly activated by a mixture of Na+ and K+. The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL beta-lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases.
Chemotherapy-induced neuropathic pain is a distressing and commonly occurring side effect of many commonly used chemotherapeutic agents, which in some cases may prevent cancer patients from being able to complete their treatment. Cannabinoid based therapies have the potential to manage or even prevent pain associated with this syndrome. Pre-clinical animal studies that investigate the modulation of the endocannabinoid system (endogenous cannabinoid pathway) are being conducted to better understand the mechanisms behind this phenomenon. Five recent pre-clinical studies identified from Medline published between 2013 and 2016 were selected for review. All studies evaluated the effect of small-molecule agonists or antagonists on components of the endocannabinoid system in rats or mice, using cisplatin or paclitax-el-induced allodynia as a model of chemotherapy-induced neuropathic pain. Activation of the cannabinoid receptor-2 (CB-2) receptor by AM1710 blocked paclitaxel-induced mechanical and cold allodynia in one study. Four studies investigating the activation of both cannabinoid receptor-1 (CB-1) and CB-2 receptors by dual-agonists (WIN55,21 and CP55,940), or by the introduction of inhibitors of endocannabinoid metabolisers (URB597, URB937, JZL184, and SA-57) showed reduction of chemotherapy-induced al-lodynia. In addition, their results suggest that anti-allodynic effects may also be mediated by additional receptors, including TRPV1 and 5-hydroxytryptamine (5-HT1A). Pre-clinical studies demon-strate that the activation of endocannabinoid CB-1 or CB-2 receptors produces physiological effects in animal models, namely the reduction of chemotherapy-induced allodynia. These studies also provide in-sight into the biological mechanism behind the therapeutic utility of cannabis compounds in managing chemotherapy-induced neuropathic pain, and provide a basis for the conduct of future clinical studies in patients of this population.
A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100 degrees C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30 degrees C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions.
Title: Saturation mutagenesis in selected amino acids to shift Pseudomonas sp. acidic lipase Lip I.3 substrate specificity and activity Panizza P, Cesarini S, Diaz P, Rodriguez Giordano S Ref: Chem Commun (Camb), 51:1330, 2015 : PubMed
Several Pseudomonas sp. CR611 Lip I.3 mutants with overall increased activity and a shift towards longer chain substrates were constructed. Substitution of residues Y29 and W310 by smaller amino acids provided increased activity on C18-substrates. Residues G152 and S154, modified to study their influence on interfacial activation, displayed a five and eleven fold increased activity.
Title: Unusual carboxylesterase bearing a GGG(A)X-type oxyanion hole discovered in Paenibacillus barcinonensis BP-23 Infanzon B, Valenzuela SV, Fillat A, Pastor FI, Diaz P Ref: Biochimie, 104:108, 2014 : PubMed
Strain Paenibacillus barcinonensis BP-23, previously isolated from Ebro's river delta (Spain), bears a complex hydrolytic system showing the presence of at least two enzymes with activity on lipidic substrates. EstA, a cell-bound B-type carboxylesterase from the strain was previously isolated and characterized. The gene coding for a second putative lipase, located upstream cellulase Cel5A, was obtained using a genome walking strategy and cloned in Escherichia coli for further characterization. The recombinant clone obtained displayed high activity on medium/short-chain fatty acid-derivative substrates. The enzyme, named Est23, was purified and characterized, showing maximum activity on pNP-caprylate (C8:0) or MUF-heptanoate (C7:0) under conditions of moderate temperature and pH. Although Est23 displays a GGG(A)X-type oxyanion hole, described as an important motif for tertiary alcohol ester resolution, neither conversion nor enantiomeric resolution of tertiary alcohols could be detected. Amino acid sequence alignment of Est23 with those of known bacterial lipase families and with closely related proteins suggests that the cloned enzyme does not belong to any of the described bacterial lipase families. A phylogenetic tree including Est23 and similar amino acid sequences showed that the enzyme belongs to a differentiated sequence cluster which probably constitutes a new family of bacterial lipolytic enzymes.
Title: Special Rhodococcus sp. CR-53 esterase Est4 contains a GGG(A)X-oxyanion hole conferring activity for the kinetic resolution of tertiary alcohols Bassegoda A, Fillat A, Pastor FI, Diaz P Ref: Applied Microbiology & Biotechnology, 97:8559, 2013 : PubMed
Rhodococci are highly adaptable bacteria, capable to degrade or transform a large number of organic compounds, including recalcitrant or toxic products. However, little information is available on the lipases of the genus Rhodococcus, except for LipR, the first lipase isolated and described from strain Rhodococcus CR-53. Taking into consideration the interest raised by the enzymes produced by actinomycetes, a search for new putative lipases was performed in strain Rhodococcus CR-53. We describe here the isolation, cloning, and characterization of intracellular esterase Est4, a mesophilic enzyme showing preference for short-chain-length acyl groups, without interfacial activation. Est4 displays moderate thermal and pH stability and low tolerance to most tested ions, being inhibited by detergents like sodium dodecyl sulfate and Triton X-100(R). Nevertheless, the enzyme shows good long-term stability when stored at 4-20 degrees C and neutral pH. Amino acid sequence analysis of Est4 revealed a protein of 313 amino acids without a signal peptide, bearing most of the conserved blocks that define bacterial lipase family IV, thus being assigned to this family. Detection of a GGG(A)X oxyanion hole in the enzyme motivated the evaluation of Est4 ability to convert tertiary alcohol esters. The newly discovered esterase Est4 from Rhodococcus CR-53 successfully hydrolyzed the tertiary alcohol esters linalyl acetate, terpinyl acetate, and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate.
Title: Acidic lipase Lip I.3 from a Pseudomonas fluorescens-like strain displays unusual properties and shows activity on secondary alcohols Panizza P, Syfantou N, Pastor FI, Rodriguez S, Diaz P Ref: J Appl Microbiol, 114:722, 2013 : PubMed
AIMS: Identification, cloning, expression and characterization of a novel lipase--Lip I.3--from strain Pseudomonas CR-611. METHODS AND RESULTS: The corresponding gene was identified and isolated by PCR-amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0-1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C-terminal type I secretion signal and several putative Ca(2+) binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca(2+) ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols. CONCLUSIONS: A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts. SIGNIFICANCE AND IMPACT OF THE STUDY: The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis.
Title: Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan Bassegoda A, Pastor FI, Diaz P Ref: Applied Environmental Microbiology, 78:1724, 2012 : PubMed
Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases.
Title: Differential behaviour of Pseudomonas sp. 42A2 LipC, a lipase showing greater versatility than its counterpart LipA Bofill C, Prim N, Mormeneo M, Manresa A, Pastor FI, Diaz P Ref: Biochimie, 92:307, 2010 : PubMed
Growth of Pseudomonas sp. 42A2 on oleic acid releases polymerized hydroxy-fatty acids as a result of several enzymatic conversions that could involve one or more lipases. To test this hypothesis, the lipolytic system of strain Pseudomonas sp. 42A2 was analyzed, revealing the presence of at least an intracellular carboxylesterase and a secreted lipase. Consensus primers derived from a conserved region of bacterial lipase subfamilies I.1 and I.2 allowed isolation of two secreted lipase genes, lipA and lipC, highly homologous to those of Pseudomonas aeruginosa PAO1. Homologous cloning of the isolated lipA and lipC genes was performed in Pseudomonas sp. 42A2 for LipA and LipC over-expression. The overproduced lipases were further purified and characterized, both showing preference for medium fatty acid chain-length substrates. However, significant differences could be detected between LipA and LipC in terms of enzyme kinetics and behaviour pattern. Accordingly, LipA showed maximum activity at moderate temperatures, and displayed a typical Michaelis-Menten kinetics. On the contrary, LipC was more active at low temperatures and displayed partial interfacial activation, showing a shift in substrate specificity when assayed at different temperatures, and displaying increased activity in the presence of certain heavy metal ions. The versatile properties shown by LipC suggest that this lipase could be expressed in response to variable environmental conditions.
The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.
Title: Efficient secretion of Bacillus subtilis lipase A in Saccharomyces cerevisiae by translational fusion to the Pir4 cell wall protein Mormeneo M, Andres I, Bofill C, Diaz P, Zueco J Ref: Applied Microbiology & Biotechnology, 80:437, 2008 : PubMed
Both the secretion and the cell surface display of Bacillus subtilis lipase A (Lip A) in Saccharomyces cerevisiae was investigated using different domains of the cell wall protein Pir4 as translational fusion partners. LipA gene minus its leader peptide was fused inframe in two places of PIR4 to achieve cell wall targeting, or substituting most of the PIR4 sequence, after the signal peptide and the Kex2 processed subunit I of Pir4 to achieve secretion to the growth medium. Expression of the recombinant fusion proteins was investigated in a standard and a glycosylation-deficient strain of S. cerevisiae, grown in selective or rich medium. Fusion proteins intended to be retained at the cell wall were secreted to the growth medium, most likely as result of the degradation of the Pir4 moiety containing the cell wall retention domain, giving low levels of lipase activity. However, the fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 400 IU of lipase activity per milliliter of cell supernatant. This is, to our knowledge, the first report of the efficient production, as a secreted protein, of lipase A of B. subtilis in baker's yeast.
Title: Helicobacter pylori EstV: identification, cloning, and characterization of the first lipase isolated from an epsilon-proteobacterium Ruiz C, Falcocchio S, Pastor FI, Saso L, Diaz P Ref: Applied Environmental Microbiology, 73:2423, 2007 : PubMed
Bacterial lipases are attracting an enormous amount of attention due to their wide biotechnological applications and due to their roles as virulence factors in some bacteria. Helicobacter pylori is a significant and widespread pathogen which produces a lipase(s) and phospholipases that seem to play a role in mucus degradation and the release of proinflammatory and cytotoxic compounds. However, no H. pylori lipase(s) has been isolated and described previously. Therefore, a search for putative lipase-encoding genes was performed by comparing the amino acid sequences of 53 known lipolytic enzymes with the deduced proteome of H. pylori. As a result, we isolated, cloned, purified, and characterized EstV, a novel lipolytic enzyme encoded by open reading frame HP0739 of H. pylori 26695, and classified it in family V of the bacterial lipases. This enzyme has the properties of a small, cell-bound carboxylesterase (EC 3.1.1.1) that is active mostly with short-chain substrates and does not exhibit interfacial activation. EstV is stable and does not require additional cofactors, and the maximum activity occurs at 50 degrees C and pH 10. This unique enzyme is the first lipase isolated from H. pylori that has been described, and it might contribute to ulcer development, as inhibition by two antiulcer substances (beta-aescin and glycyrrhizic acid) suggests. EstV is also the first lipase from an epsilon-proteobacterium to be described. Furthermore, this enzyme is a new member of family V, probably the least-known family of bacterial lipases, and the first lipase of this family for which kinetic behavior, inhibition by natural substances, and other key biochemical features are reported.
Title: Esterase EstA6 from Pseudomonas sp. CR-611 is a novel member in the utmost conserved cluster of family VI bacterial lipolytic enzymes Prim N, Bofill C, Pastor FI, Diaz P Ref: Biochimie, 88:859, 2006 : PubMed
Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved beta-/gamma-protobacterial cluster in family VI lipases, to which EstA6 belongs.
Title: Atomic force microscope studies on the interactions of Candida rugosa lipase and supported lipidic bilayers Prim N, Iversen L, Diaz P, Bjornholm T Ref: Colloids Surf B Biointerfaces, 52:138, 2006 : PubMed
Using the Langmuir-Blodgett technique we prepared substrate supported well-defined lipid/phospholipid (1-mono-palmitoyl-rac-glycerol (MPG)/l,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC)) bilayers in which the MPG lipid leaflet was exposed to the aqueous phase. Hydrolysis of MPG performed by Candida rugosa lipase (CRL) on the upper MPG layer of these supported bilayers on mica was imaged by real time atomic force microscope (AFM) using a liquid cell, so that the area increase of the initial structural defects could be followed over time. Our data strongly suggest that the edges of the initial structural defects are the preferred activation sites for CRL once the enzyme is adsorbed onto these interfaces. When a 2.5nM bulk concentration of CRL was assayed on this planar lipid substrate, we found a long lag phase before a sharp increase of catalytic activity. The lag-burst kinetic behaviour was related to the interfacial activation phenomenon although we propose that it is also dependent on the gel-phase state of this interface.
Subtropical soil microbial isolates were screened for carbohydrate, tributyrin, or olive oil hydrolysis using agar plates supplemented with the corresponding substrates. A heterotrophic, aerobic, Gram-positive strain displaying activity on tributyrin was selected and further characterized. Analysis of the morphological and physiological traits of the strain placed it as a member of the genus Rhodococcus. Further 16S rDNA sequencing revealed a 99% identity to Rhodococcus erythropolis. The strain displayed lipolytic activity on fatty-acid-derivative substrates of short chain length, with cell extract fractions having highest activity, as confirmed by the presence, after zymogram analysis, of a ca. 60-kDa intracellular protein band with activity on 4-methylumbelliferone-butyrate substrate. The presence of such a lipolytic enzyme, similar to those found in other Gram-positive bacteria, indicates that the strain could be of interest for certain biotechnological applications, like the synthesis of pharmaceuticals or biocide detoxification.
Title: Isolation of lipid- and polysaccharide-degrading micro-organisms from subtropical forest soil, and analysis of lipolytic strain Bacillus sp. CR-179 Ruiz C, Pastor FI, Diaz P Ref: Lett Appl Microbiol, 40:218, 2005 : PubMed
AIMS: To isolate the micro-organisms from three soil samples obtained from a subtropical forest of Puerto Iguazu (Argentina), to analyse them for detection of the biotechnologically interesting enzymatic activities lipase, esterase, cellulase, xylanase and pectinase, and to identify the most active strain. METHODS AND RESULTS: A total of 724 strains were isolated using different culture media and temperatures, and 449 of them showed at least one of the hydrolytic activities pursued. Lipolytic activity of the lipid-degrading strains was further determined using MUF-butyrate and MUF-oleate as substrates. The alkalophilic strain CR-179, one of the most active for all the enzymatic activities assayed, was characterized and preliminarily identified by morphological, physiological and 16S rDNA tests, as a Bacillus sp. closely related to Bacillus subtilis. CONCLUSIONS: Highly hydrolytic strains were isolated from all soil samples, suggesting the existence of a microbial community well-adapted to nutrient recycling. Strain CR-179, one of the most active, has been preliminarily identified as a Bacillus sp. SIGNIFICANCE AND IMPACT OF THE STUDY: A collection of hydrolytic strains with high biotechnological potential was obtained. Presence of sequences codifying for a lipolytic system related to the B. subtilis group lipases was revealed by PCR for the best lipolytic strain.
Title: Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay Ruiz C, Falcocchio S, Xoxi E, Pastor FI, Diaz P, Saso L Ref: Biochimica & Biophysica Acta, 1672:184, 2004 : PubMed
Research on lipase inhibitors could help in the therapy of diseases caused by lipase-producing microorganisms and in the design of novel lipase substrate specificities for biotechnology. Here we report a fast and sensitive colorimetric microassay that is low-cost and suitable for high-throughput experiments for the evaluation of lipase activity and inhibition. Comparison of Candida rugosa activity and inhibition with previous HPLC results validated the method, and revealed the importance of the reaction mixture composition. The assay was used to evaluate the effect of saturated fatty acids on Bacillus-related lipases. Cell-bound esterases were strongly inhibited by fatty acids, suggesting a negative feedback regulation by product, and a role of these enzymes in cell membrane turnover. Bacillus subtilis LipA was moderately activated by low concentrations of fatty acids and was inhibited at greater concentrations. LipB-like esterases were highly activated by myristic and lauric acids and were only slightly inhibited by high capric acid concentrations. Such an activation, reported here for the first time in bacterial lipases, seems to be part of a regulatory system evolved to ensure a high use of carbon sources, and could be related to the successful adaptation of Bacillus strains to nutrient-rich environments with strong microbial competition.
Title: Isolation and characterization of Bacillus sp. BP-6 LipA, a ubiquitous lipase among mesophilic Bacillus species Ruiz C, Javier Pastor FI, Diaz P Ref: Lett Appl Microbiol, 37:354, 2003 : PubMed
AIMS: The aim of this study was to perform the isolation, cloning and characterization of a lipase from Bacillus sp. BP-6 bearing the features of a biotechnologically important group of enzymes. METHODS AND RESULTS: Strain Bacillus sp. BP-6, showing activity on tributyrin plates, was used for isolation of lipase-coding gene lipA by means of inverse and direct PCR. The complete 633 nucleotide ORF isolated was cloned in Escherichia coli for further characterization. The amino acid sequence of the cloned protein was 98% identical to B. subtilis and B. megaterium lipases, the enzyme also showing similar molecular and biochemical features. CONCLUSIONS: The gene coding for Bacillus sp. BP-6 LipA was found in all mesophilic Bacillus species assayed, indicating its ubiquity in the genus. The cloned enzyme displayed the same properties as those of homologous lipases. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall profile of Bacillus sp. BP-6 LipA was found to be that of a ubiquitous and highly conserved subfamily I.4 bacterial lipase. Previously described lipases within this family have shown to be well suited for biotechnological applications, suggesting that the cloned enzyme could be used accordingly.
Title: Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis Lipase A Sanchez M, Prim N, Randez-Gil F, Pastor FI, Diaz P Ref: Biotechnol Bioeng, 78:339, 2002 : PubMed
Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes. The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S. cerevisiae strains. An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive.
Title: Cloning and characterization of a bacterial cell-bound type B carboxylesterase from Bacillus sp. BP-7 Prim N, Pastor FI, Diaz P Ref: Curr Microbiol, 42:237, 2001 : PubMed
A clone producing halos on tributyrin plates was isolated from a genomic library of Bacillus sp. BP-7. The insert contained an open reading frame that coded for a protein of 487 amino acids with homology to carboxylesterases. The cloned enzyme showed clear preference for esters of short-chain fatty acids, being classified as an esterase. Maximum activity was found at 45 degrees C and pH 7.5. The enzyme displayed stability in the pH range from 6 to 9.5, and at temperatures from 4 degrees to 45 degrees C. Zymogram analysis of the protein revealed a molecular mass of 53 kDa and a pI of 5.1. The enzyme showed homology to members of the bacterial subclass of type B carboxylesterases, a set of proteins potentially useful for biotechnological applications.
Title: estA, a gene coding for a cell-bound esterase from Paenibacillus sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases Prim N, Blanco A, Martinez J, Pastor FIJ, Diaz P Ref: Res Microbiol, 151:303, 2000 : PubMed
Screening of a gene library from Paenibacillus sp. BP-23 generated in Escherichia coli led to identification of a clone that directed the production of lipolytic activity. From the sequencing data, we found an open reading frame encoding a protein of 485 amino acids with an estimated molecular mass of 53 kDa and a pI of 5.1. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-XI-S-X2-G included in most serine-esterases and lipases. The cloned protein showed high homology with enzymes belonging to the bacterial subclass of type B carboxylesterases. The enzyme had a significant preference for esters of short-chain fatty acids and showed the kinetics behaviour of a true esterase. Maximum activity was found at pH 7.5 and 37 degrees C, although the enzyme was active in the pH range 6.0- 9.0 and at temperatures up to 45 degrees C. As expected for a serine-esterase, activity was inhibited by phenylmethylsulphonyl fluoride.
