Shaw JF

References (36)

Title : Isolation, Expression and Characterization of the Thermophilic Recombinant Esterase from Geobacillus thermodenitrificans PS01 - Chen_2020_Appl.Biochem.Biotechnol_191_112
Author(s) : Chen PT , Liu CH , Chen YT , Hsu FY , Shaw JF
Ref : Appl Biochem Biotechnol , 191 :112 , 2020
Abstract : Esterases are widely used in the food industry. Here, a new thermophilic bacterium, Geobacillus thermodenitrificans PS01, was isolated and the esterase-encoding gene est1 was cloned, sequenced, and recombinant expressed in Escherichia coli Tuner (DE3). The highest activity of recombinant Est1 was detected at pH 8.0, and 40 degrees C and the extreme stability was observed at pH 6-9 over 30 days at 4 degrees C. In particular, Est1 can hydrolyze short- to medium-chain (C2-C10) triglycerides and p-nitrophenyl esters (C2-C12) and was not inhibited by most metal ions. Kinetic parameters of p-nitrophenyl butyrate hydrolysis under optimal conditions were determined: Km, 22.76 muM; kcat, 10,415 s(-1); and kcat/Km, 457.53 muM(-1) s(-1). The outstanding specification of Est1 indicates its potential for use in industrial applications.
ESTHER : Chen_2020_Appl.Biochem.Biotechnol_191_112
PubMedSearch : Chen_2020_Appl.Biochem.Biotechnol_191_112
PubMedID: 31956956
Gene_locus related to this paper: geotn-a4isp3

Title : A novel recombinant chlorophyllase from cyanobacterium Cyanothece sp. ATCC 51142 for the production of bacteriochlorophyllide a - Chou_2016_Biotechnol.Appl.Biochem_63_371
Author(s) : Chou YL , Lee YL , Yen CC , Chen LF , Lee LC , Shaw JF
Ref : Biotechnol Appl Biochem , 63 :371 , 2016
Abstract : Bacteriopheophorbide a (BPheid a) is used as a precursor for bacteriochlorin a (BCA), which can be used for photodynamic therapy in both in vitro and in vivo biochemical applications. This study successfully isolated and expressed a photosynthetic bacterium (Cyanothece sp. ATCC 51142) chlorophyllase called CyanoCLH, which can be used as a biocatalyst for the production of a BCA precursor by degrading bacteriochlorophyll a (BChl a). Substrate specificity and enzyme kinetic analyses were performed and the results verified that the recombinant CyanoCLH preferred hydrolyzing BChl a to produce bacteriochlorophyllide a (BChlide a), which can be converted to BPheid a by removing magnesium ion. The recombinant CyanoCLH was cloned and expressed in Escherichia coli BL-21 (DE3), and its molecular weight was 54.7 kDa. The deduced amino acid sequence of the recombinant CyanoCLH comprised a unique lipase-motif GHSLG, which differs from the GHSRG sequence of other plants and lacks a histidine of the typical and conserved catalytic triad Ser-Asp-His. The recombinant CyanoCLH was subjected to biochemical analyses, and the results indicated that its optimal pH and temperature were 7.0 and 60 degrees C, respectively.
ESTHER : Chou_2016_Biotechnol.Appl.Biochem_63_371
PubMedSearch : Chou_2016_Biotechnol.Appl.Biochem_63_371
PubMedID: 25828734

Title : Conversion of crude Jatropha curcas seed oil into biodiesel using liquid recombinant Candida rugosa lipase isozymes - Kuo_2015_Bioresour.Technol_192_54
Author(s) : Kuo TC , Shaw JF , Lee GC
Ref : Bioresour Technol , 192 :54 , 2015
Abstract : The versatile Candida rugosa lipase (CRL) has been widely used in biotechnological applications. However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isozymes. In the present study, four liquid recombinant CRL isozymes (CRL1-CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50wt% water, an initial 1equivalent of methanol feeding, and an additional 0.5equivalents of methanol feeding at 24h for a total reaction time of 48h at 37 degrees C). We concluded that specific recombinant CRL isozymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil.
ESTHER : Kuo_2015_Bioresour.Technol_192_54
PubMedSearch : Kuo_2015_Bioresour.Technol_192_54
PubMedID: 26011691

Title : A Novel Recombinant Chlorophyllase1 from Chlamydomonas reinhardtii for the Production of Chlorophyllide Derivatives - Chou_2015_J.Agric.Food.Chem_63_9496
Author(s) : Chou YL , Ko CY , Yen CC , Chen LF , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 63 :9496 , 2015
Abstract : Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 degreesC, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce chlorophyllide derivatives.
ESTHER : Chou_2015_J.Agric.Food.Chem_63_9496
PubMedSearch : Chou_2015_J.Agric.Food.Chem_63_9496
PubMedID: 26478543
Gene_locus related to this paper: chlre-a8j2s9

Title : Simultaneous production of fatty acid methyl esters and diglycerides by four recombinant Candida rugosa lipase's isozymes - Chang_2014_Food.Chem_155_140
Author(s) : Chang SW , Huang M , Hsieh YH , Luo YT , Wu TT , Tsai CW , Chen CS , Shaw JF
Ref : Food Chem , 155 :140 , 2014
Abstract : In this study, the catalytic efficiency of four recombinant CRL (Candida rugosa lipase) isozymes (LIP1-LIP4) towards the production of fatty acid methyl ester (FAME) was compared and evaluated as an alternative green method for industrial applications. The results indicated that the recombinant C. rugosa LIP1 enzyme exhibited the highest catalytic efficiency for FAME production compared to the recombinant C. rugosa LIP2-LIP4 enzymes. The optimal conditions were as follows: pH 7.0, methanol/soybean oil molar ratio: 3/1, enzyme amount: 2U (1.6muL), reaction temperature: 20 degrees C, 22h of reaction time, and 3 times of methanol addition (1mol/6h), and resulted in 61.5+/-1.5wt.% of FAME conversion. The reaction product contained also 10wt.% of DAG with a ratio of 1,3-DAG to 1,2-DAG of approximately 4:6, and can be potentially used in industrial applications as a food emulsifier.
ESTHER : Chang_2014_Food.Chem_155_140
PubMedSearch : Chang_2014_Food.Chem_155_140
PubMedID: 24594166

Title : Multifunctionality and diversity of GDSL esterase\/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis - Chepyshko_2012_BMC.Genomics_13_309
Author(s) : Chepyshko H , Lai CP , Huang LM , Liu JH , Shaw JF
Ref : BMC Genomics , 13 :309 , 2012
Abstract : BACKGROUND: GDSL esterases/lipases are a newly discovered subclass of lipolytic enzymes that are very important and attractive research subjects because of their multifunctional properties, such as broad substrate specificity and regiospecificity. Compared with the current knowledge regarding these enzymes in bacteria, our understanding of the plant GDSL enzymes is very limited, although the GDSL gene family in plant species include numerous members in many fully sequenced plant genomes. Only two genes from a large rice GDSL esterase/lipase gene family were previously characterised, and the majority of the members remain unknown. In the present study, we describe the rice OsGELP (Oryza sativa GDSL esterase/lipase protein) gene family at the genomic and proteomic levels, and use this knowledge to provide insights into the multifunctionality of the rice OsGELP enzymes. RESULTS: In this study, an extensive bioinformatics analysis identified 114 genes in the rice OsGELP gene family. A complete overview of this family in rice is presented, including the chromosome locations, gene structures, phylogeny, and protein motifs. Among the OsGELPs and the plant GDSL esterase/lipase proteins of known functions, 41 motifs were found that represent the core secondary structure elements or appear specifically in different phylogenetic subclades. The specification and distribution of identified putative conserved clade-common and -specific peptide motifs, and their location on the predicted protein three dimensional structure may possibly signify their functional roles. Potentially important regions for substrate specificity are highlighted, in accordance with protein three-dimensional model and location of the phylogenetic specific conserved motifs. The differential expression of some representative genes were confirmed by quantitative real-time PCR. The phylogenetic analysis, together with protein motif architectures, and the expression profiling were analysed to predict the possible biological functions of the rice OsGELP genes. CONCLUSIONS: Our current genomic analysis, for the first time, presents fundamental information on the organization of the rice OsGELP gene family. With combination of the genomic, phylogenetic, microarray expression, protein motif distribution, and protein structure analyses, we were able to create supported basis for the functional prediction of many members in the rice GDSL esterase/lipase family. The present study provides a platform for the selection of candidate genes for further detailed functional study.
ESTHER : Chepyshko_2012_BMC.Genomics_13_309
PubMedSearch : Chepyshko_2012_BMC.Genomics_13_309
PubMedID: 22793791

Title : Engineering the expression and biochemical characteristics of recombinant Candida rugosa LIP2 lipase by removing the additional N-terminal peptide and regional codon optimization - Chang_2011_J.Agric.Food.Chem_59_6710
Author(s) : Chang SW , Li CF , Lee GC , Yeh T , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 59 :6710 , 2011
Abstract : Candida rugosa lipase (CRL), an important industrial enzyme, has been established, containing several different isoforms which were encoded by the high-identity lip gene family (lip1 to lip7). In this study, we compared the expression and biochemical characterization with three different engineered lip2 constructions in the yeast Pichia pastoris. Our results showed that lip2 (lip2) has an overall improvement of 50% higher production yield (1.446 U/mL) relative to that of nflip2 (0.964 U/mL) at 7 days of cultivation time. Codon-optimized lip2 (colip2) has a 2.3-fold higher production yield (2.182 U/mL) compared to that of lip2 (noncodon-optimized; 1.446 U/mL) and nflip2 (0.964 U/mL), with a cultivation time of 5 days. This finding demonstrated that the removal of the N-terminus and the regional codon optimization of the lip2 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP2 in the P. pastoris system. The distinct biochemical properties of our purified recombinant nfLIP2 and LIP2 suggested that they are potentially useful for various industrial applications.
ESTHER : Chang_2011_J.Agric.Food.Chem_59_6710
PubMedSearch : Chang_2011_J.Agric.Food.Chem_59_6710
PubMedID: 21561168

Title : Characterization of codon-optimized recombinant candida rugosa lipase 5 (LIP5) - Lee_2011_J.Agric.Food.Chem_59_10693
Author(s) : Lee LC , Yen CC , Malmis CC , Chen LF , Chen JC , Lee GC , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 59 :10693 , 2011
Abstract : Recombinant Candida rugosa lipase 5 (LIP5) has been functionally expressed along with other isoforms in our laboratory. However, the characterization and codon optimization of LIP5 have not been done. In this work, we characterized, codon-optimized and compared LIP5 with commercial lipase. LIP5 activity on hydrolysis of p-nitrophenyl (p-NP) butyrate was optimal at 55 degrees C as compared with 37 degrees C of the commercial lipase. Several assays were also performed to determine the substrate specificity of LIP5. p-NP butyrate (C(4)), butyryl-CoA (C(4)), cholesteryl laurate (C(12)), and N-carbobenzoxy-l-tyrosine-p-nitrophenyl ester (l-NBTNPE) were found as preferred substrates of LIP5. Interestingly, LIP5 specificity on hydrolysis of amino acid-derivative substrates was shown to be the highest among any lipase isoforms, but it had very weak preference on hydrolyzing triacylglycerol substrates. LIP5 also displays a pH-dependent maximum activity of a lipase but an esterase substrate preference in general. The characterization of LIP5 along with that of LIP1-LIP4 previously identified shows that each lipase isoform has a distinct substrate preference and catalytic activity.
ESTHER : Lee_2011_J.Agric.Food.Chem_59_10693
PubMedSearch : Lee_2011_J.Agric.Food.Chem_59_10693
PubMedID: 21854055

Title : Functional proteomic analysis of rice bran esterases\/lipases and characterization of a novel recombinant esterase - Chuang_2011_J.Agric.Food.Chem_59_2019
Author(s) : Chuang HH , Chen PT , Wang WN , Chen YT , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 59 :2019 , 2011
Abstract : An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 degrees C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 muM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) muM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.
ESTHER : Chuang_2011_J.Agric.Food.Chem_59_2019
PubMedSearch : Chuang_2011_J.Agric.Food.Chem_59_2019
PubMedID: 21322560

Title : Site-specific saturation mutagenesis on residues 132 and 450 of Candida rugosa LIP2 enhances catalytic efficiency and alters substrate specificity in various chain lengths of triglycerides and esters - Yen_2010_J.Agric.Food.Chem_58_10899
Author(s) : Yen CC , Malmis CC , Lee GC , Lee LC , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 58 :10899 , 2010
Abstract : The catalytic versatility of recombinant Candida rugosa LIP2 has been known to have potential applications in industry. In this study, site-specific saturation mutagenesis on residues L132 and G450 of recombinant LIP2 has been employed to investigate the impact of both residues on substrate specificity of LIP2. Point mutations on L132 and G450 were done separately using mutagenic degenerate primer sets containing 32 codons to generate two libraries of mutants in Pichia pastoris . Replacements of amino acid on these mutants were identified as L132A, L132I, G450S, and G450A. In lipase activity assay, L132A and L132I mutants showed a shift of preference from short- to medium-chain triglyceride, whereas G450S and G450A mutants retained preferences as compared to wild-type LIP2. Among mutants, G450A has the highest activity on tributyrin. However, hydrolysis of p-nitrophenyl (p-NP) esters with L132A, L132I, and G450S did not show differences of preferences over medium- to long-chain esters except in G450A, which prefers only medium-chain ester as compared to wild-type LIP2. All mutants showed an enhanced catalytic activity and higher optimal temperature and pH stability as compared to wild-type LIP2.
ESTHER : Yen_2010_J.Agric.Food.Chem_58_10899
PubMedSearch : Yen_2010_J.Agric.Food.Chem_58_10899
PubMedID: 20873770

Title : Genes and biochemical characterization of three novel chlorophyllase isozymes from Brassica oleracea - Lee_2010_J.Agric.Food.Chem_58_8651
Author(s) : Lee GC , Chepyshko H , Chen HH , Chu CC , Chou YF , Akoh CC , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 58 :8651 , 2010
Abstract : Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.
ESTHER : Lee_2010_J.Agric.Food.Chem_58_8651
PubMedSearch : Lee_2010_J.Agric.Food.Chem_58_8651
PubMedID: 20681655

Title : Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomea - Hsu_2010_J.Appl.Microbiol_108_1712
Author(s) : Hsu KH , Wang SY , Chu FH , Shaw JF
Ref : J Appl Microbiol , 108 :1712 , 2010
Abstract : AIMS: A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized. METHODS AND RESULTS: The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 -bp open reading frame encoding 314 amino acids with a molecular weight of 35.5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser-His-Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C-terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long-chain acyl esterases at pH 7 and 30 degrees C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4 degrees C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry. CONCLUSION: We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level.
ESTHER : Hsu_2010_J.Appl.Microbiol_108_1712
PubMedSearch : Hsu_2010_J.Appl.Microbiol_108_1712
PubMedID: 19849771
Gene_locus related to this paper: taica-d2t083

Title : Structure of the alkalohyperthermophilic Archaeoglobus fulgidus lipase contains a unique C-terminal domain essential for long-chain substrate binding - Chen_2009_J.Mol.Biol_390_672
Author(s) : Chen CK , Lee GC , Ko TP , Guo RT , Huang LM , Liu HJ , Ho YF , Shaw JF , Wang AH
Ref : Journal of Molecular Biology , 390 :672 , 2009
Abstract : Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 A resolution. This enzyme has optimal activity in the temperature range of 70-90 degrees C and pH 10-11. AFL consists of an N-terminal alpha/beta-hydrolase fold domain, a small lid domain, and a C-terminal beta-barrel domain. The N-terminal catalytic domain consists of a 6-stranded beta-sheet flanked by seven alpha-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a beta-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.
ESTHER : Chen_2009_J.Mol.Biol_390_672
PubMedSearch : Chen_2009_J.Mol.Biol_390_672
PubMedID: 19447113
Gene_locus related to this paper: arcfu-AF1763

Title : Promoter analysis and differential expression of the Candida rugosa lipase gene family in response to culture conditions - Hsu_2008_J.Agric.Food.Chem_56_1992
Author(s) : Hsu KH , Lee GC , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 56 :1992 , 2008
Abstract : Five lipase genes have been identified and sequenced from Candida rugosa. However, as the sequences of LIP multigene family are extremely closely related, it is difficult to characterize the expression spectrum of LIP genes. In the present work we have cloned, sequenced, and analyzed the promoters of these five LIP isoform genes, and several putative transcriptional elements including oleate response element (ORE) and upstream activation sequence 1 (UAS1) were identified. A quantitative real-time RT-PCR method was developed for determining the differential expression of C. rugosa lipase family genes in response to various environmental and nutritional factors. While all five LIP genes display significant changes in mRNA expression under oleic acid and/or olive oil culture conditions, LIP2 showed the strongest induction (456-fold) in response to oleic acid. LIP transcription and promoter regulation were studied by assaying the beta-galactosidase activities of promoter-lacZ fusions in Saccharomyces cerevisiae. Three of the LIP genes, LIP3, LIP4, and LIP5, showed significant induction by oleic acid, and their ORE and UAS1 elements are essential for induction by oleic acid. Together, this suggests that the multiple lipase expression profiles may be due to differential transcriptional regulation of the LIP genes in response to environment or nutritional factors.
ESTHER : Hsu_2008_J.Agric.Food.Chem_56_1992
PubMedSearch : Hsu_2008_J.Agric.Food.Chem_56_1992
PubMedID: 18290622

Title : Transgenic broccoli (Brassica oleracea var. italica) with antisense chlorophyllase (BoCLH1) delays postharvest yellowing. - Chen_2008_Plant.Sci_174_25
Author(s) : Chen LFO , Lin CH , Kelkar SM , Chang YM , Shaw JF
Ref : Plant Sci , 174 :25 , 2008
Abstract : Postharvest yellowing in broccoli is known to result from chlorophyll degradation, with chlorophyllase the first enzyme to degrade chlorophyll. In broccoli, three putative chlorophyllase genes (BoCLH1, BoCLH2, and BoCLH3) were cloned using degenerate primers from the conserved regions of known chlorophyllases. Among these three genes, only BoCLH1 is transcribed during the course of broccoli postharvest senescence. A chimeric construct with the antisense BoCLH1, driven by the CaMV 35S promoter and Nos-terminator and harboring the hygromycin resistance gene, was used for Agrobacterium-mediated transformation to study the effects of the antisense BoCLH1 gene on the postharvest senescence of broccoli. From a total of about 90 primary transformants, 35 individuals were selected and grown for further studies, with 22 of these grown to maturity. Based on the Chl retention rate on Days 4 and 5, respectively, 45% of the detached florets and over 60% of the detached leaves of the selected transformants exhibited slower yellowing when stored at 20C in darkness. Southern blot analyses were conducted to eliminate the possible non-independent transformants and investigate the insertion patterns and copy numbers. Only a few lines with simple insertion site and copy number and postharvest yellowing retardation effects were self-pollintated for further evaluation. Northern analyses showed antisense BoCLH1 mRNA transcripts on the day of harvest, the levels of which gradually decreased 4-5 days postharvest when stored at 20C in darkness. Positive correlations between the antisense BoCLH1 transcripts and slower postharvest yellowing were noted in some selected lines. Only 1-2 days delay in yellowing was observed in the selected antisense BoCLH1-positive transformants. No individual antisense BoCLH2 or BoCLH3 transformants showed significant slowing of postharvest yellowing. The results suggested genes other than the BoCLH-Chlases obtained in the present study might also be essential in the yellowing process.
ESTHER : Chen_2008_Plant.Sci_174_25
PubMedSearch : Chen_2008_Plant.Sci_174_25
PubMedID:
Gene_locus related to this paper: braol-Q8GTM3

Title : Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly(gamma-glutamic acid) by RSM - Chang_2008_Bioresour.Technol_99_2800
Author(s) : Chang SW , Shaw JF , Yang KH , Chang SF , Shieh CJ
Ref : Bioresour Technol , 99 :2800 , 2008
Abstract : Poly(gamma-glutamic acid) (gamma-PGA) is a material of polymer. Immobilization of Candida rugosa lipase (Lipase AY-30) by covalent binding on gamma-PGA led to a markedly improved performance of the enzyme. Response surface methodology (RSM) and 3-level-3-factor fractional factorial design were employed to evaluate the effects of immobilization parameters, such as immobilization time (2-6h), immobilization temperature (0-26 degrees C), and enzyme/support ratio (0.1-0.5, w/w). Based on the analysis of ridge max, the optimum immobilization conditions were as follows: immobilization time 2.3h, immobilization temperature 13.3 degrees C, and enzyme/support ratio 0.41 (w/w); the highest lipase activity obtained was 1196 U/mg-protein.
ESTHER : Chang_2008_Bioresour.Technol_99_2800
PubMedSearch : Chang_2008_Bioresour.Technol_99_2800
PubMedID: 17706421

Title : Identification and characterization of a lipase gene from Antrodia cinnamomea - Chu_2008_Mycol.Res_112_1421
Author(s) : Chu FH , Wang SY , Lee LC , Shaw JF
Ref : Mycol Res , 112 :1421 , 2008
Abstract : A partial (634 bp) cDNA clone, AF1229, obtained from expressed sequence tags (ESTs) of solid-cultured basidiomes of Antrodia cinnamomea is homologous to the lipase gene in Rhizomucor miehei. 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE amplification showed that the full-length lipase gene, Ac-LIP, has a 912bp open reading frame (ORF), a 183bp 5' non-coding region, and a 144bp 3' non-coding region. Ac-LIP contains the lipase consensus sequence, VTVVGHSLGA, and encodes a 303-amino acid polypeptide that appears to be an extracellular protein with a calculated molecular mass of 31.8 kDa. RT-PCR analysis suggested that Ac-LIP was strongly expressed during the basidiomatal formation stage of A. cinnamomea. When over-expressed in Escherichia coli, Ac-LIP yielded a protein that was capable of performing hydrolysis of trilinolein by gas chromatography/mass spectrometry (GC/MS) analysis. A. cinnamomea lipase represents the first enzyme of the lipase family from a basidiomycetous fungus, which has been characterized at the molecular level.
ESTHER : Chu_2008_Mycol.Res_112_1421
PubMedSearch : Chu_2008_Mycol.Res_112_1421
PubMedID: 18652894
Gene_locus related to this paper: taica-a8qm10

Title : Synthesis and characterization of canola oil-stearic acid-based trans-free structured lipids for possible margarine application - Lumor_2007_J.Agric.Food.Chem_55_10692
Author(s) : Lumor SE , Jones KC , Ashby R , Strahan GD , Kim BH , Lee GC , Shaw JF , Kays SE , Chang SW , Foglia TA , Akoh CC
Ref : Journal of Agricultural and Food Chemistry , 55 :10692 , 2007
Abstract : Incorporation of stearic acid into canola oil to produce trans-free structured lipid (SL) as a healthy alternative to partially hydrogenated fats for margarine formulation was investigated. Response surface methodology was used to study the effects of lipozyme RM IM from Rhizomucor miehei and Candida rugosa lipase isoform 1 (LIP1) and two acyl donors, stearic acid and ethyl stearate, on the incorporation. Lipozyme RM IM and ethyl stearate gave the best result. Gram quantities of SLs were synthesized using lipozyme RM IM, and the products were compared to SL made by chemical catalysis and fat from commercial margarines. After short-path distillation, the products were characterized by GC and RPHPLC-MS to obtain fatty acid and triacylglycerol profiles, 13C NMR spectrometry for regiospecific analysis, X-ray diffraction for crystal forms, and DSC for melting profile. Stearic acid was incorporated into canola oil, mainly at the sn-1,3 positions, for the lipase reaction, and no new trans fatty acids formed. Most SL products did not have adequate solid fat content or beta' crystal forms for tub margarine, although these may be suitable for light margarine formulation.
ESTHER : Lumor_2007_J.Agric.Food.Chem_55_10692
PubMedSearch : Lumor_2007_J.Agric.Food.Chem_55_10692
PubMedID: 18052237

Title : Candida rugosa lipase LIP1-catalyzed transesterification to produce human milk fat substitute - Srivastava_2006_J.Agric.Food.Chem_54_5175
Author(s) : Srivastava A , Akoh CC , Chang SW , Lee GC , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 54 :5175 , 2006
Abstract : Structured lipids (SLs) containing palmitic and oleic acids were synthesized by transesterification of tripalmitin with either oleic acid or methyl oleate as acyl donor. This SL with palmitic acid at the sn-2 position and oleic acid at sn-1,3 positions is similar in structure to human milk fat triacylglycerol. LIP1, an isoform of Candida rugosa lipase (CRL), was used as biocatalyst. The effects of reaction temperature, substrate molar ratio, and time on incorporation of oleic acid were investigated. Reaction time and temperature were set at 6, 12, and 24 h, and 35, 45, and 55 degrees C, respectively. Substrate molar ratio was varied from 1:1 to 1:4. The highest incorporation of oleic acid (37.7%) was at 45 degrees C with methyl oleate as acyl donor. Oleic acid resulted in slightly lesser (26.3%) incorporation. Generally, higher percentage incorporation of oleic acid was observed with methyl oleate (transesterification) than with oleic acid (acidolysis). In both cases percentage incorporation increased with reaction time. Incorporation decreased with increase in temperature above 45 degrees C. Initially, oleic acid incorporation increased with increase in substrate molar ratio up to 1:3. LIP1 was also compared with Lipozyme RM IM as biocatalysts. The tested reaction parameters were selected on the basis of maximum incorporation of C18:1 obtained during optimization of LIP1 reaction conditions. Reaction temperature was maintained at 45, 55, and 65 degrees C. Lipozyme RM IM gave highest oleic acid incorporation (49.4%) at 65 degrees C with methyl oleate as acyl donor. Statistically significant (P < 0.05) differences were observed for both enzymes. SL prepared using Lipozyme RM IM may be more suitable for possible use in human milk fat substitutes.
ESTHER : Srivastava_2006_J.Agric.Food.Chem_54_5175
PubMedSearch : Srivastava_2006_J.Agric.Food.Chem_54_5175
PubMedID: 16819932

Title : Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase - Chang_2006_J.Agric.Food.Chem_54_815
Author(s) : Chang SW , Lee GC , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 54 :815 , 2006
Abstract : An important industrial enzyme, Candida rugosa lipase (CRL) possesses several different isoforms encoded by the lip gene family (lip1-lip7), in which the recombinant LIP1 is the major form of the CRL multigene family. Previously, 19 of the nonuniversal serine codons (CTG) of the lip1 gene hav been successfully converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP1 in the yeast Pichia pastoris. To improve the expression efficiency of recombinant LIP1 in P. pastoris, a regional synthetic gene fragment of lip1 near the 5' end of a transcript has been constructed to match P. pastoris-preferred codon usage for simple scale-up fermentation. The present results show that the production level (152 mg/L) of coLIP1 (codon-optimized LIP1) has an overall improvement of 4.6-fold relative to that (33 mg/L) of non-codon-optimized LIP1 with only half the cultivation time of P. pastoris. This finding demonstrates that the regional codon optimization the lip1 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP1 in the P. pastoris system. More distinct biochemical properties of the purified recombinant LIP1 for further industrial applications are also determined and discussed in detail.
ESTHER : Chang_2006_J.Agric.Food.Chem_54_815
PubMedSearch : Chang_2006_J.Agric.Food.Chem_54_815
PubMedID: 16448188

Title : Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris - Chang_2005_Appl.Microbiol.Biotechnol_67_215
Author(s) : Chang SW , Shieh CJ , Lee GC , Shaw JF
Ref : Applied Microbiology & Biotechnology , 67 :215 , 2005
Abstract : Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization. In this study, we utilized PCR to remove an unnecessary linker of pGAPZalphaC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 in the Pichia expression system. Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6-38.4 degrees C), glucose concentration (0.3-3.7%), yeast extract (0.16-1.84%), and pH (5.3-8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris. Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1 degrees C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6. The predicted value of lipolytic activity was 246.9+/-39.7 U/ml, and the actual value was 253.3+/-18.8 U/ml. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.
ESTHER : Chang_2005_Appl.Microbiol.Biotechnol_67_215
PubMedSearch : Chang_2005_Appl.Microbiol.Biotechnol_67_215
PubMedID: 15592826

Title : A fine physical map of the rice chromosome 5 - Cheng_2005_Mol.Genet.Genomics_274_337
Author(s) : Cheng CH , Chung MC , Liu SM , Chen SK , Kao FY , Lin SJ , Hsiao SH , Tseng IC , Hsing YI , Wu HP , Chen CS , Shaw JF , Wu J , Matsumoto T , Sasaki T , Chen HH , Chow TY
Ref : Mol Genet Genomics , 274 :337 , 2005
Abstract : A fine physical map of the rice (Oryza sativa spp. Japonica var. Nipponbare) chromosome 5 with bacterial artificial chromosome (BAC) and PI-derived artificial chromosome (PAC) clones was constructed through integration of 280 sequenced BAC/PAC clones and 232 sequence tagged site/expressed sequence tag markers with the use of fingerprinted contig data of the Nipponbare genome. This map consists of five contigs covering 99% of the estimated chromosome size (30.08 Mb). The four physical gaps were estimated at 30 and 20 kb for gaps 1-3 and gap 4, respectively. We have submitted 42.2-Mb sequences with 29.8 Mb of nonoverlapping sequences to public databases. BAC clones corresponding to telomere and centromere regions were confirmed by BAC-fluorescence in situ hybridization (FISH) on a pachytene chromosome. The genetically centromeric region at 54.6 cM was covered by a minimum tiling path spanning 2.1 Mb with no physical gaps. The precise position of the centromere was revealed by using three overlapping BAC/PACs for approximately 150 kb. In addition, FISH results revealed uneven chromatin condensation around the centromeric region at the pachytene stage. This map is of use for positional cloning and further characterization of the rice functional genomics.
ESTHER : Cheng_2005_Mol.Genet.Genomics_274_337
PubMedSearch : Cheng_2005_Mol.Genet.Genomics_274_337
PubMedID: 16261349
Gene_locus related to this paper: orysa-gid1 , orysa-q6f358 , orysj-PLA7

Title : GDSL family of serine esterases\/lipases - Akoh_2004_Prog.Lipid.Res_43_534
Author(s) : Akoh CC , Lee GC , Liaw YC , Huang TH , Shaw JF
Ref : Prog Lipid Res , 43 :534 , 2004
Abstract : GDSL esterases and lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity. They have potential for use in the hydrolysis and synthesis of important ester compounds of pharmaceutical, food, biochemical, and biological interests. This new subclass of lipolytic enzymes possesses a distinct GDSL sequence motif different from the GxSxG motif found in many lipases. Unlike the common lipases, GDSL enzymes do not have the so called nucleophile elbow. Studies show that GDSL hydrolases have a flexible active site that appears to change conformation with the presence and binding of the different substrates, much like the induced fit mechanism proposed by Koshland. Some of the GDSL enzymes have thioesterase, protease, arylesterase, and lysophospholipase activity, yet they appear to be the same protein with similar molecular weight ( approximately 22-60 kDa for most esterases), although some have multiple glycosylation sites with higher apparent molecular weight. GDSL enzymes have five consensus sequence (I-V) and four invariant important catalytic residues Ser, Gly, Asn, and His in blocks I, II, III, and V, respectively. The oxyanion structure led to a new designation of these enzymes as SGNH-hydrolase superfamily or subfamily. Phylogenetic analysis revealed that block IIA which belonged to the SGNH-hydrolases was found only in clade I. Therefore, this family of hydrolases represents a new example of convergent evolution of lipolytic enzymes. These enzymes have little sequence homology to true lipases. Another important differentiating feature of GDSL subfamily of lipolytic enzymes is that the serine-containing motif is closer to the N-terminus unlike other lipases where the GxSxG motif is near the center. Since the first classification of these subclass or subfamily of lipases as GDSL(S) hydrolase, progress has been made in determining the consensus sequence, crystal structure, active site and oxyanion residues, secondary structure, mechanism of catalysis, and understanding the conformational changes. Nevertheless, much still needs to be done to gain better understanding of in vivo biological function, 3-D structure, how this group of enzymes evolved to utilize many different substrates, and the mechanism of reactions. Protein engineering is needed to improve the substrate specificity, enantioselectivity, specific activity, thermostability, and heterologous expression in other hosts (especially food grade microorganisms) leading to eventual large scale production and applications. We hope that this review will rekindle interest among researchers and the industry to study and find uses for these unique enzymes.
ESTHER : Akoh_2004_Prog.Lipid.Res_43_534
PubMedSearch : Akoh_2004_Prog.Lipid.Res_43_534
PubMedID: 15522763

Title : Protein engineering and applications of Candida rugosa lipase isoforms - Akoh_2004_Lipids_39_513
Author(s) : Akoh CC , Lee GC , Shaw JF
Ref : Lipids , 39 :513 , 2004
Abstract : Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.
ESTHER : Akoh_2004_Lipids_39_513
PubMedSearch : Akoh_2004_Lipids_39_513
PubMedID: 15554150

Title : Sequential structural changes of Escherichia coli thioesterase\/protease I in the serial formation of Michaelis and tetrahedral complexes with diethyl p-nitrophenyl phosphate - Tyukhtenko_2003_Biochemistry_42_8289
Author(s) : Tyukhtenko SI , Litvinchuk AV , Chang CF , Lo YC , Lee SJ , Shaw JF , Liaw YC , Huang TH
Ref : Biochemistry , 42 :8289 , 2003
Abstract : Escherichia coli thioesterase/protease I (TEP-I) belongs to a new subclass of lipolytic enzymes of the serine hydrolase superfamily. Here we report the first direct NMR observation of the formation of the Michaelis complex (MC) between TEP-I and diethyl p-nitrophenyl phosphate (DENP), an active site directed inhibitor of serine protease, and its subsequent conversion to the tetrahedral complex (TC). NMR, ESI-MS, and kinetic data showed that DENP binds to TEP-I in a two-step process, a fast formation of MC followed by a slow conversion to TC. NMR chemical shift perturbation further revealed that perturbations were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. The largest chemical shift perturbation occurred around the catalytic Ser(10). In MC, the conformation of the mobile Ser(10) was stabilized, and its amide resonance became observable. From the large chemical shift perturbation upon conversion from MC to TC, we propose that the amide protons of Ser(10) and Gly(44) serve as the oxyanion hole proton donors that stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP. The present study also demonstrates the important catalytic roles of conserved residues in the SGNH family of proteins.
ESTHER : Tyukhtenko_2003_Biochemistry_42_8289
PubMedSearch : Tyukhtenko_2003_Biochemistry_42_8289
PubMedID: 12846577

Title : Crystal structure of Escherichia coli thioesterase I\/protease I\/lysophospholipase L1: consensus sequence blocks constitute the catalytic center of SGNH-hydrolases through a conserved hydrogen bond network - Lo_2003_J.Mol.Biol_330_539
Author(s) : Lo YC , Lin SC , Shaw JF , Liaw YC
Ref : Journal of Molecular Biology , 330 :539 , 2003
Abstract : Escherichia coli thioesterase I (TAP) is a multifunctional enzyme possessing activities of thioesterase, esterase, arylesterase, protease, and lysophospholipase. In particular, TAP has stereoselectivity for amino acid derivative substrates, hence it is useful for the kinetic resolution of racemic mixtures of industrial chemicals. In the present work, the crystal structure of native TAP was determined at 1.9A, revealing a minimal SGNH-hydrolase fold. The structure of TAP in complex with a diethyl phosphono moiety (DEP) identified its catalytic triad, Ser10-Asp154-His157, and oxyanion hole, Ser10-Gly44-Asn73. The oxyanion hole of TAP consists of three residues each separated from the other by more than 3.5A, implying that all of them are highly polarized when substrate bound. The catalytic (His)C(epsilon1)-H...O=C hydrogen bond usually plays a role in the catalytic mechanisms of most serine hydrolases, however, there were none present in SGNH-hydrolases. We propose that the existence of the highly polarized tri-residue-constituted oxyanion hole compensates for the lack of a (His)C(epsilon1)-H...O=C hydrogen bond. This suggests that members of the SGNH-hydrolase family may employ a unique catalytic mechanism. In addition, most SGNH-hydrolases have low sequence identities and presently there is no clear criterion to define consensus sequence blocks. Through comparison of TAP and the three SGNH-hydrolase structures currently known, we have identified a unique hydrogen bond network which stabilizes the catalytic center: a newly discovered structural feature of SGNH-hydrolases. We have defined these consensus sequence blocks providing a basis for the sub-classification of SGNH-hydrolases.
ESTHER : Lo_2003_J.Mol.Biol_330_539
PubMedSearch : Lo_2003_J.Mol.Biol_330_539
PubMedID: 12842470

Title : NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase\/protease I - Tyukhtenko_2002_FEBS.Lett_528_203
Author(s) : Tyukhtenko SI , Litvinchuk AV , Chang CF , Leu RJ , Shaw JF , Huang TH
Ref : FEBS Letters , 528 :203 , 2002
Abstract : Escherichia coli thioesterase/protease I (TEP-I) is a lipolytic enzyme of the serine protease superfamily with Ser(10), Asp(154) and His(157) as the catalytic triad residues. Based on comparison of the low-field (1)H nuclear magnetic resonance spectra of two mutants (S10G and S12G) and two transition state analogue complexes we have assigned the exchangeable proton resonances at 16.3 ppm, 14.3 ppm, and 12.8 ppm at pH 3.5 to His(157)-N(delta1)H, Ser(10)-O(gamma)H and His(157)-N(epsilon2)H, respectively. Thus, the presence of a strong Asp(154)-His(157) hydrogen bond in free TEP-I was observed. However, Ser(10)-O(gamma)H was shown to form a H-bond with a residue other than His(157)-N(epsilon2).
ESTHER : Tyukhtenko_2002_FEBS.Lett_528_203
PubMedSearch : Tyukhtenko_2002_FEBS.Lett_528_203
PubMedID: 12297305

Title : Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris - Lee_2002_Biochem.J_366_603
Author(s) : Lee GC , Lee LC , Sava V , Shaw JF
Ref : Biochemical Journal , 366 :603 , 2002
Abstract : The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH 7 and a broad temperature optimum in the range 30-50 degrees C. The enzyme retained 80% of residual activity after being heated at 70 degrees C for 10 min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12-C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2.
ESTHER : Lee_2002_Biochem.J_366_603
PubMedSearch : Lee_2002_Biochem.J_366_603
PubMedID: 12020350

Title : Factors affecting the resolution of dl-menthol by immobilized lipase-catalyzed esterification in organic solvent - Wang_2002_J.Agric.Food.Chem_50_262
Author(s) : Wang DL , Nag A , Lee GC , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 50 :262 , 2002
Abstract : Among 10 lipases tested, Candida rugosa lipase exhibited the best ability to catalyze the resolution of dl-menthol in organic solvent. The lipase was immobilized on different carriers, and the experiment was carried out with different acyl donors. The high yield and optical purity of the product were achieved in cyclohexane with valeric acid as acyl donor using C. rugosa lipase immobilized on DEAE-Sephadex A-25. The conversion of dl-menthol depended on the water content of immobilized lipase and on the pH of the aqueous solution from which lipase was immobilized. The operational stability of the DEAE-Sephadex A-25 immobilized lipase in catalysis of the esterification reaction showed that >85% activity remained after 34 days of repeated use. The resolution of racemic menthol in organic medium catalyzed by immobilized C. rugosa lipase-catalyzed esterification is very convenient, and it represents a significant improvement in the use of enzyme for the preparative production of optically active menthol. This process is readily applicable to large-scale preparation.
ESTHER : Wang_2002_J.Agric.Food.Chem_50_262
PubMedSearch : Wang_2002_J.Agric.Food.Chem_50_262
PubMedID: 11782192

Title : Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability - Tang_2001_Arch.Biochem.Biophys_387_93
Author(s) : Tang SJ , Shaw JF , Sun KH , Sun GH , Chang TY , Lin CK , Lo YC , Lee GC
Ref : Archives of Biochemistry & Biophysics , 387 :93 , 2001
Abstract : Although Candida rugosa utilizes a nonuniversal serine codon (CUG) for leucine, it is possible to express lipase genes (LIP) in heterologous systems. After replacing the 19 CUG codons in LIP4 with serine codons by site-directed mutagenesis, a recombinant LIP4 was functionally overexpressed in Pichia pastoris in this study. This recombinant glycosylated lipase was secreted into the culture medium with a high purity of 100 mg/liter in a culture broth. Purified recombinant LIP4 had a molecular mass of 60 kDa, showing a range similar to that of lipase in a commercial preparation. Since LIP4 has only a glycosylation site at position Asn-351, this position may also be the major glycosylation site in C. rugosa lipases. Although the thermal stability of recombinant LIP4 significantly increased from 52 to 58 degrees C after glycosylation, there were no significant differences in the catalytic properties of recombinant glycosylated lipase from P. pastoris and the unglycosylated one from Escherichia coil. These two recombinant LIP4s showed higher esterase activities toward long-chain ester (C16 and C18) and exhibited higher lipase activities toward unsaturated and long-chain lipids. In addition, LIP4 does not show interfacial activation as compared with LIP1 toward lipid substrates of tributyrin and triolein. These observations demonstrated that LIP4 shows distinguished catalytic activities with LIP1 in spite of their high sequence homology.
ESTHER : Tang_2001_Arch.Biochem.Biophys_387_93
PubMedSearch : Tang_2001_Arch.Biochem.Biophys_387_93
PubMedID: 11368188

Title : Synthesis of fatty acid esters by recombinant Staphylococcus epidermidis lipases in aqueous environment - Chang_2001_J.Agric.Food.Chem_49_2619
Author(s) : Chang RC , Chou SJ , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 49 :2619 , 2001
Abstract : Various flavor esters were obtained by using recombinant lipases from Staphylococcus epidermidis as a catalyst in an aqueous environment. These esters were enzymatically synthesized to overcome the problems associated with chemical processes. This study showed that the S. epidermidis lipases could catalyze ester synthesis from decyl alcohol and fatty acids of different chain length. The wild-type and mutant lipases (M419A and V649I) could efficiently catalyze the synthesis of decyl alcohol esters of unsaturated fatty acids. In contrast, the yield of decyl laurate was better by wild-type and mutant enzyme V6491, but mutant enzyme M419A only favored the synthesis of decyl myristate. The esterification of oleic acid and various carbon-chain-length alcohols from ethanol to hexadecanol increased up to decanol by wild-type and M419A mutant enzymes and reached an optimum for dodecanol by V6491 mutant enzyme. The enzyme is potentially useful in food industries such as dairy product flavoring.
ESTHER : Chang_2001_J.Agric.Food.Chem_49_2619
PubMedSearch : Chang_2001_J.Agric.Food.Chem_49_2619
PubMedID: 11368644

Title : Lipase-catalyzed synthesis of fatty acid diethanolamides - Liu_2001_J.Agric.Food.Chem_49_5761
Author(s) : Liu KJ , Nag A , Shaw JF
Ref : Journal of Agricultural and Food Chemistry , 49 :5761 , 2001
Abstract : Diethanolamides are nonionic emulsifiers widely used in industries such as cosmetics and as corrosion inhibitors. Candida antarctica lipase (Novozym 435) was used to catalyze the amidation of various fatty acids with diethanolamine. Contents of fatty acids, metal ions, and water affected the yields of diethanolamides. Hexanoic acid was the best substrate among all acyl donors. Yields of hexanoyl diethanolamide (HADEA), lauroyl diethanolamide (LADEA), and oleoyl diethanolamide (OADEA), obtained after 24 h of lipase-catalyzed reaction at 50 degrees C and 250 rpm with 90 mM fatty acid and 360 mM diethanolamine in acetonitrile, were 76.5, 49.5, and 12.1%, respectively. Addition of 1 mM metal salts increased the yields of HADEA and LADEA. Kinetic analysis showed that the yields of HADEA and LADEA in lipase-catalyzed reactions were largely associated with the rate of the forward reaction constant k(1). Anhydrous enzyme was found to be the best for the amidation reaction. Study on the enzyme operational stability showed that C. antarctica lipase retained 95 and 85% of the initial activity for the syntheses of HADEA and LADEA, respectively (even after repeated use for 10 days). The reaction runs smoothly without the use of hazardous reactants, and the developed method is useful for the industrial application.
ESTHER : Liu_2001_J.Agric.Food.Chem_49_5761
PubMedSearch : Liu_2001_J.Agric.Food.Chem_49_5761
PubMedID: 11743760

Title : Crystallization and preliminary X-ray crystallographic analysis of thioesterase I from Escherichia coli - Lo_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_756
Author(s) : Lo YC , Lee YL , Shaw JF , Liaw YC
Ref : Acta Crystallographica D Biol Crystallogr , 56 :756 , 2000
Abstract : The Escherichia coli thioesterase I specifically catalyzes the deacylation of fatty acyl-CoA thioesters, especially those with long acyl groups (C(12)-C(18)). Single crystals of thioesterase I (E.C. 3. 1.2.2) from E. coli have been obtained using methoxypolyethylene glycol 5000 (PEG-MME 5K) as a precipitant at room temperature in 21 d. The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2, with unit-cell parameters a = b = 50.85 (7), c = 171.5 (1) A. The crystals diffract to beyond 2.4 A resolution. There is one molecule of molecular weight 20.5 kDa in the asymmetric unit, with a solvent content of 55%.
ESTHER : Lo_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_756
PubMedSearch : Lo_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_756
PubMedID: 10818355

Title : Multinuclear NMR resonance assignments and the secondary structure of Escherichia coli thioesterase\/protease I: a member of a new subclass of lipolytic enzymes - Lin_1998_J.Biomol.NMR_11_363
Author(s) : Lin TH , Chen C , Huang RF , Lee YL , Shaw JF , Huang TH
Ref : J Biomol NMR , 11 :363 , 1998
Abstract : Escherichia coli thioesterase/protease I is a 183 amino acid protein with a molecular mass of 20,500. This protein belongs to a new subclass of lipolytic enzymes of the serine protease superfamily, but with a new GDSLS consensus motif, of which no structure has yet been determined. The protein forms a tetramer at pH values above 6.5 and exists as a monomer at lower pH values. Both monomer and tetramer are catalytically active. From analysis of a set of heteronuclear multidimensional NMR spectra with uniform and specific amino acid labeled protein samples, we have obtained near-complete resonance assignments of the backbone 1H, 13C and 15N nuclei (BMRB databank accession number 4060). The secondary structure of E. coli thioesterase/protease I was further deduced from the consensus chemical shift indices, backbone short- and medium-range NOEs, and amide proton exchange rates. The protein was found to consist of four beta-strands and seven alpha-helices, arranged in alternate order. The four beta-strands were shown to form a parallel beta-sheet. The topological arrangement of the beta-strands of -1x, +2x, +1x appears to resemble that of the core region of the alpha beta hydrolase superfamily, typically found in common lipases and esterases. However, substantial differences, such as the number of beta-strands and the location of the catalytic triad residues, make it difficult to give a definitive classification of the structure of E. coli thioesterase/protease I at present.
ESTHER : Lin_1998_J.Biomol.NMR_11_363
PubMedSearch : Lin_1998_J.Biomol.NMR_11_363
PubMedID: 9691282

Title : The thioesterase I of Escherichia coli has arylesterase activity and shows stereospecificity for protease substrates - Lee_1997_Biochem.Biophys.Res.Commun_231_452
Author(s) : Lee YL , Chen JC , Shaw JF
Ref : Biochemical & Biophysical Research Communications , 231 :452 , 1997
Abstract : A thioesterase I gene was recloned and sequenced from Escherichia coli strain JM109. The overexpressed, matured enzyme from JM109 was purified to homogeneity. The enzyme showed broad hydrolytic activity toward three kinds of substrates including acyl-CoAs, esters, and amino acid derivatives. The enzyme had a kcat/Km value of 0.363 s-1 microM-1, for a typical thioesterase I substrate, palmitoyl-CoA. The arylesterase activity of the enzyme was observed by its ability to hydrolyze several aromatic esters including alpha-naphthyl acetate, alpha-naphthyl butyrate, phenyl acetate, benzyl acetate, and eight p-nitrophenyl esters. In kinetic studies a chymotrypsin-like substrate (an amino acid derivative), N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (L-NBPNPE), was the best substrate for the enzyme with a catalytic efficiency (kcat/Km) of 4.00 s-1 microM-1, which was 23 times higher than that of the enantiomer D-NBPNPE (0.171 s-1 microM-1). It was concluded that the thioesterase I of E. coli had arylesterase activity and it possessed stereospecificity for protease substrates.
ESTHER : Lee_1997_Biochem.Biophys.Res.Commun_231_452
PubMedSearch : Lee_1997_Biochem.Biophys.Res.Commun_231_452
PubMedID: 9070299

Title : Facile purification of highly active recombinant Staphylococcus hyicus lipase fragment and characterization of a putative lid region - Chang_1996_Biochem.Biophys.Res.Commun_228_774
Author(s) : Chang RC , Chen JC , Shaw JF
Ref : Biochemical & Biophysical Research Communications , 228 :774 , 1996
Abstract : A fragment of Staphylococcus hyicus lipase gene (Ala248 to Ala640) was inserted into plasmid pET20(b+). The resulting His-tagged recombinant protein (49 kDa) was overexpressed in Escherichia coli BL21(DE3) as an highly active lipase and was purified by nickel-coupled resin. Site-directed mutagenesis showed that in comparison with wild type enzyme, the L326F and L326A enzymes showed a 3.4 and 5 fold increase in the K(m), respectively, but only a 44% and a 64% decrease in the kcat/K(m), respectively, suggesting that Leu326 of the putative lid participated in substrate-binding.
ESTHER : Chang_1996_Biochem.Biophys.Res.Commun_228_774
PubMedSearch : Chang_1996_Biochem.Biophys.Res.Commun_228_774
PubMedID: 8941352
Gene_locus related to this paper: stahy-lipas