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Ergosterol (Ergo) and cholesterol contribute to performances of liposomes by increasing membrane packing density and physical stability. However, as these sterols can reduce membrane flexibility, they can lower skin permeability of liposomes. We synthesized ergosterol ester (Ergo-Est) containing unsaturated fatty acid different from Ergo in size and physical properties. In this work, we investigated effects of Ergo-Est and Ergo on physical properties of liposomes. We incorporated Ergo, Ergo-oleate (EO

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.

Vanillyl alcohol (VA), which is abundant in Vanilla bean, has strong antioxidant activity. However, the use of VA in the food and cosmetics industries is limited, due to its low solubility in emulsion or organic solvents. Meanwhile, medium chain fatty acids and medium chain monoglycerides have antibacterial activity. We synthesized butyric acid vanillyl ester (BAVE) or caprylic acid vanillyl ester (CAVE) from VA with tributyrin or tricaprylin through transesterification reaction using immobilized lipases. BAVE and CAVE scavenged 2,2-diphenyl-1-picrylhydrazyl radicals in organic solvents. In addition, BAVE and CAVE decreased the production rate of conjugated diene and triene in the menhaden oil-in-water emulsion system. While BAVE showed no antibacterial activity, CAVE showed antibacterial activity against food spoilage bacteria, including Bacillus coagulans. In this study, the antibacterial activity of vanillyl ester with medium chain fatty acid was first revealed. Zeta potential measurements confirmed that BAVE and CAVE were inserted into B. coagulans membrane. In addition, the propidium iodide uptake assay and fluorescent microscopy showed that CAVE increased B. coagulans membrane permeability. Therefore, CAVE is expected to play an important role in the food and cosmetics industries as a bi-functional material with both antioxidant and antibacterial activities.

An esterase gene, estA1, was cloned from Alteromonas sp. 39-G1 isolated from the Beaufort Sea. The gene is composed of 1,140 nucleotides and codes for a 41,190 Da protein containing 379 amino acids. As a result of a BLAST search, the protein sequence of esterase EstA1 was found to be identical to Alteromonas sp. esterase (GenBank: PHS53692). As far as we know, no research on this enzyme has yet been conducted. Phylogenetic analysis showed that esterase EstA1 was a member of the bacterial lipolytic enzyme family IV (hormone sensitive lipases). Two deletion mutants (Δ20 and Δ54) of the esterase EstA1 were produced in Escherichia coli BL21 (DE3) cells with part of the N-terminal of the protein removed and His-tag attached to the C-terminal. These enzymes exhibited the highest activity toward p-nitrophenyl (pNP) acetate (C2) and had little or no activity towards pNP-esters with acyl chains longer than C6. Their optimum temperature and pH of the catalytic activity were 45°C and pH 8.0, respectively. As the NaCl concentration increased, their enzyme activities continued to increase and the highest enzyme activities were measured in 5 M NaCl. These enzymes were found to be stable for up to 8 h in the concentration of 3-5 M NaCl. Moreover, they have been found to be stable for various metal ions, detergents and organic solvents. These salt-tolerant and chemical-resistant properties suggest that the enzyme esterase EstA1 is both academically and industrially useful.

Microbial lipases are used widely in the synthesis of various compounds due to their substrate specificity and position specificity. 4-Ethyl malate (4-EM) made from diethyl malate (DEM) is an important starting material used to make argon fluoride (ArF) photoresist. We tested several microbial lipases and found that

Several phenolic compounds derived from plant biomass are attracting attention because they display high antioxidant activity. In this study, antioxidant activity was confirmed in 4-hydroxyphenylacetic acid (HPA), and transesterification reaction using Candida antarctica lipase B was performed to enhance the solubility of HPA. The HPA-diolein (HPA-DON) was synthesized from HPA and triolein, while the HPA-fish oil diglyceride (HPA-dFO) was synthesized from HPA and Menhaden fish oil. To increase the conversion yield, the enzyme reaction conditions of the substrate molar ratio, enzyme amount, and reaction time were optimized. After the reaction, HPA-DON and HPA-dFO were purely separated, using prep-LC. The activity assays using DPPH and ABTS radicals confirmed that HPA-DON and HPA-dFO have antioxidant activity. HPA-DON has high activity in non-polar solvents, while HPA-dFO has strong activity in both polar solvents and non-polar solvents. In addition, HPA-dFO has the growth inhibition activity for Bacillus coagulans, Geobacillus stearothermophilus, and Alcaligenes faecalis that cause food spoilage. Therefore, HPA-dFO is a new synthetic substance that has both antioxidant activity and antibacterial activity. The results indicate that these HPA-derivatives can be expected to be developed as important materials in the food and cosmetics industries.

A lipolytic enzyme (Rcut) was discovered from the Rhodococcusstrain (RosL12) isolated from the Antarctic Ross Sea. The corresponding gene composed of 651 bases encoding 216 amino acids. It was found to be a cutinase gene through BLAST search. Rcut has a signal sequence consisting of 29 amino acids. An active Rcut was produced after the intact gene containing the signal sequence was transformed into Escherichia coli Rosetta-gami™ 2 (DE3) pLysS. Rcut was purified through a nickel-nitrilotriacetic acid purification system and a carboxymethyl Sepharose column chromatography. Its specific activity was 2190 U/mg. Rcut showed the highest activity at 40 °C and had a low activation energy of 3.16 kcal/mol. This means that it is a typical cold-adapted enzyme. Rcut showed high activity towards medium chain fatty acids (C

Ergosterol, like cholesterol, has many beneficial physiological activities, and because it has no adverse clinical problem with cholesterol, it is a substance that can be used as a biomaterial in the food and cosmetics industry. However, ergosterol has low oil solubility and is easily crystallized, which is problematic for its direct use in the industry. This problem can be solved by combining fatty acids with ergosterol. In this study, ergosterol derivatives with unsaturated fatty acids were synthesized from ergosterol and various plant oils. Specifically, ergosterol oleate (EO), ergosterol linoleate (EL), and ergosterol linolenate (ELn) were synthesized using Proteus vulgaris K80 lipase. To effectively synthesize these unsaturated fatty acid ergosterol esters (FAEEs), 25 mM ergosterol and 40 mM plant oil were added into hexane solvent, and transesterification reaction was performed at 40 °C. Proteus vulgaris K80 lipase showed higher conversion yield than other commercial lipases, due to its high affinity to ergosterol and broad substrate specificity. Rapeseed oil, sunflower seed oil, and linseed oil were used to synthesize EO, EL, and ELn, respectively. The FAEEs were purified, and their purity was confirmed by FT-IR and LC–MS. The solubility of the FAEEs in a tricaprylin solvent was increased 11–16 times, compared to that of ergosterol. We found that EL- and ELn-containing emulsions had strong growth inhibitory activity against some dairy and cosmetic spoilage bacteria.

An esterase gene

Lysine decarboxylase (CadA) converts L-lysine into cadaverine (1,5-pentanediamine), which is an important platform chemical with many industrial applications. Although there have been many efforts to produce cadaverine through the soluble CadA enzyme or Escherichia coli whole cells overexpressing the CadA enzyme, there have been few reports concerning the immobilization of the CadA enzyme. Here, we have prepared a cross-linked enzyme aggregate (CLEA) of E. coli CadA and performed bioconversion using CadACLEA. CadAfree and CadACLEA were characterized for their enzymatic properties. The optimum temperatures of CadAfree and CadACLEA were 60°C and 55°C, respectively. The thermostability of CadACLEA was significantly higher than that of CadAfree. The optimum pH of both enzymes was 6.0. CadAfree could not be recovered after use, whereas CadACLEA was rapidly recovered and the residual activity was 53% after the 10th recycle. These results demonstrate that CadACLEA can be used as a potential catalyst for efficient production of cadaverine.

Vanillyl alcohol (VA) is a good antioxidant compound, but its widespread use is impeded due to its low solubility in emulsion or organic solvent systems. Furthermore, several fatty acids have antimicrobial activity, but their broad spread is limited by their susceptibility to oxidation. In this study, we carried out a Proteus vulgaris K80 lipase-mediated transesterification reaction using VA and menhaden oil (MO) to produce fatty acid vanillyl ester (FAVE) and achieved a conversion yield of 75.3%. FAVE purified by prep-HPLC showed 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and the scavenging activity increased significantly in non-polar solvent. FAVE also inhibited the formation of conjugated diene and triene in MO-in-water emulsion stabilized by Tween 20. Furthermore, FAVE showed evident antibacterial activity against typical food spoilage bacteria by interacting with cell membrane and releasing cell constituent. These results demonstrated that FAVE is a bi-functional compound showing both antioxidant and antibacterial activities and it can be used as an essential component in food and cosmetic products.

Sugar esters are valuable compounds composed of various sugars and fatty acids that can be used as antibacterial agents and emulsifiers in toothpaste and canned foods. For example, fructose fatty acid esters suppress growth of Streptococcus mutans, a typical pathogenic bacterium causing dental caries. In this study, fructose laurate ester was chosen as a target material and was synthesized by a transesterification reaction using Candida antarctica lipase B. We performed a solvent screening experiment and found that a t-butanol/dimethyl sulfoxide mixture was the best solvent to dissolve fructose and methyl laurate. Fructose laurate was synthesized by transesterification of fructose (100 mM) with methyl laurate (30 mM) in t-butanol containing 20% dimethyl sulfoxide. The conversion yield was about 90%, which was calculated based on the quantity of methyl laurate using high-performance liquid chromatography. Fructose monolaurate (Mr 361) was detected in the reaction mixture by high-resolution mass spectrometry. The inhibitory effect of fructose laurate on growth of oral or food spoilage microorganisms, including S. mutans, Bacillus coagulans, and Geobacillus stearothermophilus, was evaluated.

Natural phenolic acids, such as cinnamic acid, have antioxidant and other biological activities. However, their hydrophilic groups reduce antioxidant efficiency in fat and oil systems. To overcome this restriction in their usage, natural phenolic acids have been modified via an enzymatic transesterification reaction. The lipase-catalyzed transesterification reaction has received attention due to advantages, such as stereo and regioselectivity and substrate specificity. In this study, ethyl cinnmate (40 mM) and triolein (240 mM) were used as substrates and immobilized Proteus vulgaris K80 lipase (1 U) was used as enzyme catalyst. When the interesterification reaction was performed in 5 mL n -hexane/toluene (85:15, v/v) at 35 °C for 72 h, cinnamoyl monooleyl glycerol (CMOG) and cinnamoyl dioleyl glycerol (CDOG) were produced and the conversion yield was 70%. These cinnamoylated lipid products were collected by preparative liquid chromatography and identified using high resonance mass spectrometry. CMOG and CDOG showed radical scavenging activities of 45% and 69%, respectively, in n -hexane system using DPPH assay and 56% and 20%, respectively, in dichloromethane/DMSO (8:2, v/v) system using ABTS assay.

Castor oil extracted from castor bean has antibacterial property, and has been used in various folk remedies. The major structural component of castor oil, ricinoleic acid, has actual antibacterial activity. Some phenolic compounds derived from plants have antioxidant property. Among them, vanillyl alcohol from vanilla bean has strong antioxidant activity. As vanillyl alcohol has low solubility in hydrophobic solvents and castor oil has low solubility in hydrophilic solvents, there is practical difficulty in using them. We performed lipase-mediated transesterification using vanillyl alcohol and castor oil, and synthesized ricinoleic acid vanillyl ester (RAVE). 2,2-Diphenyl-1-picrylhydrazyl assay and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assay revealed that RAVE had a strong antioxidant activity in various organic solvents. RAVE also had antibacterial activity against some food spoilage bacteria. It showed more powerful antibacterial activity for gram positive bacteria than for gram negative bacteria. The critical micelle concentration of RAVE was measured at 7.36 μM and it partitioned exclusively into emulsion phase in water-emulsion system. Zeta potential measurement, membrane release test, and fluorescent microscopy showed that RAVE inserted itself into the bacterial cell membrane, destroyed membrane permeability, and induced cell death. As such, RAVE is a novel multi-functional compound with antioxidant and antibacterial activity, so it can be used as a functional material in the food and cosmetic industries.

The Antarctic marine environment provides a good source of novel lipolytic enzymes that possess beneficial properties, i.e., resistance to extreme physical and chemical conditions. We found a lipolytic Escherichia coli colony that was transformed using genomic DNA from Marinobacter lipolyticus 27-A9 isolated from the Antarctic Ross Sea. DNA sequence analysis revealed an open reading frame of lipolytic enzyme gene. The gene translates a protein (LipA9) of 404 amino acids with molecular mass of 45,247 Da. Recombinant LipA9 was expressed in E. coli BL21 (DE3) cells and purified by anion exchange and gel filtration chromatography. The kcat/Km of LipA9 was 175 s−1 μM−1, and the optimum temperature and pH were 70 °C and pH 8.0, respectively. LipA9 had quite high organic solvent stability; it was stable toward several common organic solvents up to 50% concentration. Substrate specificity studies showed that LipA9 preferred a short acyl chain length of p-nitrophenyl ester and triglyceride. Sequence analysis showed that LipA9 contained catalytic Ser72 and Lys75 in S-x-x-K motif, like family VIII esterases. Homology modeling and site-directed mutagenesis studies revealed that Tyr141 and Tyr188 residues were located near the conserved motif and played an important role in catalytic activity.

For the synthesis of various pharmaceuticals, chiral alcohols are useful intermediates. Among them, (R)-ethyl-4-chloro-3-hydroxybutanoate ((R)-ECHB) is an important building block for the synthesis of L-carnitine. (R)-ECHB is produced from ethyl-4-chloro-3-oxobutanoate (ECOB) by a reductase-mediated, enantioselective reduction reaction. The Saccharomyces cerevisiae YOL151W reductase that is expressed in Escherichia coli cells exhibited an enantioselective reduction reaction toward ECOB. By virtue of the C-terminal His-tag, the YOL151W reductase was purified from the cell-free extract using Ni(2+)-NTA column chromatography and immobilized onto Ni(2+)-magnetic microparticles. The physical properties of the immobilized reductase (Imm-Red) were measured using electron microscopy, a magnetic property measurement system, and a zeta potential system; the average size of the particles was approximately 1 μm and the saturated magnetic value was 31.76 emu/g. A neodymium magnet was used to recover the immobilized enzyme within 2 min. The Imm-Red showed an optimum temperature at 45°C and an optimum pH at 6.0. In addition, Bacillus megaterium glucose dehydrogenase (GDH) was produced in the E. coli cells and was used in the coupling reaction to regenerate the NADPH cofactor. The reduction/oxidation coupling reaction composed of the Imm-Red and GDH converted 20 mM ECOB exclusively into (R)- ECHB with an e.e.p value of 98%.

Lipases have been utilized industrially to produce biodiesel, oleochemicals, and pharmaceuticals. Many efforts such as metagenomics, directed evolution, and enzyme immobilization have been devoted to enhance the lipase activity. Here, we designed a recombinant lipase, NKC-M37-MAT, that was generated by incorporating an N-terminal amphipathic peptide (NKC) and a C-terminal coiled-coil peptide (MAT) into Photobacterium lipolyticum M37 lipase. The hydrophobic face of NKC improve the accessibility (Km), and catalytic efficiency (Kcat/Km) of the soluble lipase toward the hydrophobic substrate and tetrameric MAT further enhanced lipase catalytic activity (U/mg) through cooperative binding to its substrate such that the catalytic activity (U/mg) of NKC-M37-MAT was increased by a maximum of 54-fold compared with the wild-type, which decreased the biodiesel production time 5-fold from 30 to 6 h. This novel approach shows promise as a platform technology to increase lipase catalytic efficiency for industrial-scale ...

Staphylococcus haemolyticus L62 lipase is methanol-stable enzyme and its functional expression has been performed in Escherichia coli cells. The recombinant lipase L62 was immobilized on amine-magnetic microparticles (AMPs) by protein precipitation and covalent cross-linking methods. The physical properties of L62-AMP were evaluated by electron microscopy, a zeta potential system, and magnetic property measurement system. L62-AMPs had diameters of about a 1.6 μm size and displayed a saturation magnetic value of 25.56 A m2/kg. In comparison with free lipase L62, L62-AMP showed high optimum temperature and high activity at a wide pH range. L62-AMP showed high hydrolytic activity toward tributyrin and olive oil among various triglycerides. L62-AMP was recovered within 2 min with Neodymium magnet and maintained >90% residual activity after 4 recoveries. L62-AMP was used to produce fatty acid methyl ester (FAME) using methanol and olive oil with molar ratio of 6:1. By one step-transesterification reaction, the yield of FAME was about 86.4% after a 24 h reaction at 30 °C.

We investigated transesterification activity of methanol-stable lipase from Staphylococcus haemolyticus L62. Lipase L62 transesterified olive oil and menhaden oil using various alcohols including methanol, ethanol, 1-propanol, and 1-butanol. Based on the high stability of lipase L62 toward alcohols, high molar ratios (3:1 or 6:1) of alcohol to oil were employed in the reaction systems. Lipase L62 produced fatty acid esters efficiently by one-step transesterification. Reaction conditions for production of fatty acid methyl esters (FAMEs) and omega-3 fatty acid ethyl esters were further optimized. Lipase L62 produced those esters efficiently when 30–40 % water content was present in the reaction medium. The lipase L62 conversion yield was as high as 92 % for FAME and omega-3 ethyl ester production. These results indicate that lipase L62 is a suitable catalyst for producing an omega-3 ethyl ester concentrate in the pharmaceutical industry as well as for producing biodiesel in the bio-refinery industry.

Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the outer membrane of Escherichia coli using the OmpA signal peptide and the autotransporter EstAβ8 protein. Localization of SHL62 lipase on the outer membrane of E. coli was confirmed using immunofluorescence microscopy and flow cytometry analysis. Lipase activity of the displayed SHL62 lipase was also measured using spectrophotometry and pH titration. SHL62 lipase activity of whole cells reached 2.0 U/ml culture (OD 600 nm of 10) when it was measured by the p -nitrophenyl caprylate assay after being induced with 1 mM IPTG for 24 h. The optimum temperature and pH for the lipase was 45 °C and 10, respectively. Furthermore, it maintained more than 90% of maximum lipase activity at up to 50 °C and in a pH range of 5–9. The hydrolytic activity assay conduted with various substrates confirmed that p -nitrophenyl caprylate and corn oil were preferred substrates among various synthetic and natural substrates, respectively. The displayed SHL62 lipase produced fatty acid esters from various alcohols and plant oils through transesterification.

Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the cell surface of Escherichia coli using an autotransporter protein of Pseudomonas putida EstAβ8 as an anchoring motif. Localization of the SHL62 lipase on the cell surface of E. coli was confirmed by immunofluorescence microscopy, flow cytometry analysis, Western blot, protease accessibility, and whole-cell enzyme activity assays. SHL62 lipase activity of whole cells reached 168 U/g dry cell weight toward p -nitrophenyl caprylate after being induced by 0.1 mM IPTG for 24 h. The optimum temperature and pH of the displayed SHL62 lipase was 40–45 °C and pH 10, respectively. The displayed SHL62 was stable up to 45 °C, at which it had >80% of maximum activity. The displayed SHL62 lipase showed a preference for medium chain fatty acids of p -nitrophenyl esters. Moreover, the displayed SHL62 lipase was used as a whole cell catalyst to produce biodiesel that was obtained at a yield of nearly 89.4% after a 96 h reaction at 30 °C. These results suggest that the displayed SHL62 lipase on the cell surface of E. coli using an EstAβ8 anchoring motif can be used for biocatalytic applications.

carnitine, and the (S)-enantiomer is an importantstarting material for hydroxymethylglutaryl-CoA reductaseinhibitors [11, 13]. Enzymatic reduction of ethyl-4-chloro-3-oxobutanoate (ECOB) by some reductase enzymes is auseful process in the asymmetric synthesis of chiral ECHB[1, 17, 19].These enzymatic reduction reactions require a largeamount of the expensive cofactor NADPH; hence, practicalapplications require efficient recycling of the cofactor [5,16]. Generally, cofactors can be regenerated through“coupled-substrate” [14, 21] and “coupled-enzyme” [5, 11,15] approaches. The “coupled-enzyme” method is the moregeneral approach and utilizes the first enzyme for thedesired biotransformation and the second to regenerate thecofactor. Until now, some X-ray crystal structures of carbonylreductase enzymes have been elucidated and their ternarycomplexes with substrate and cofactor have been disclosed[4, 8, 10]. Based on these early X-ray structures, manyhomology and docking models for other reductases havebeen suggested [8]. Detailed structural information aboutsubstrate- and cofactor-binding sites is very important tounderstand their reaction mechanisms. In addition, newmutant enzymes can be constructed through rational designand site-directed mutagenesis to change their cofactorpreference or enhance enantioselectivity [2, 12, 23].In a previous study, we generated a variety of microbialreductases in E. coli cells [3]. Among these, the yeastreductase YOR120W converted ECOB exclusively into(R)-ECHB. In this study, we purified and characterizedYOR120W and conducted a coupling reaction with Bacillus

A bacterial strain that produces a cold-adapted esterase was isolated from tidal flats and identified as Shewanella sp. Ke75. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (957 bp) corresponded to a protein of 318 amino acid residues with a calculated molecular weight of 34875 Da. The esterase showed 68 and 57% identities with the putative esterases of Shewanella amazonensis SB2B and Colwellia psychrerythraea 34H, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein Ke75 was produced in both soluble and insoluble forms when Escherichia coli cells harboring the gene were cultured at 30°C. The enzyme showed specificity for C4 (butyrate) as a substrate, with little activity toward the other p-nitrophenyl esters tested. The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity remained up to 60% even at 5°C with an activation energy of 6.29 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was enhanced in the presence of Mn2+ ions, but inhibited by Cd2+, Cu2+, Hg2+, and Zn2+ ions.

Various plant oils and animal fats can be used for the production of biodiesel, a renewable and eco-friendly alternative fuel. In this research, transesterification of olive oil and methanol was performed using Staphylococcus haemolyticus L62 lipase expressed in Escherichia coli cells. L62 lipase was relatively methanol-stable and could produce biodiesel by a one-step methanol-feeding process. This enzyme was immobilized on a poly (methacrylate-co-divinyl benzene) resin by two different methods: hydrophobic adsorption/entrapment (H-L62) and hydrophobic adsorption/entrapment plus covalent cross-linking (HC-L62). Both immobilized enzymes showed quite increased temperature and pH stabilities, whereas they had very similar optimal temperature and pH in comparison with the soluble free enzyme. Soluble L62, H-L62 and HC-L62 lipases could produce biodiesel efficiently from olive oil. Among them, HC-L62 lipase was the most robust enzyme on the basis of multiple reusability. HC-L62 produced biodiesel with various plant oils including palm, olive fomase oils as well as waste cooking oil, suggesting that it can be utilized as a biocatalyst in the biodiesel industry.

A lipase-producing Photobacterium strain (MA1-3) was isolated from the intestine of a blood clam caught at Namhae, Korea. The lipase gene was cloned by shotgun cloning and encoded 340 amino acids with a molecular mass of 38,015 Da. It had a very low sequence identity with other bacterial lipases, with the exception of that of Photobacterium lipolyticum M37 (83.2%). The MA1-3 lipase was produced in soluble form when Escherichia coli cells harboring the gene were cultured at 18°C. Its optimum temperature and pH were 45°C and pH 8.5, respectively. Its activation energy was calculated to be 2.69 kcal/mol, suggesting it to be a cold-adapted lipase. Its optimum temperature, temperature stability, and substrate specificity were quite different from those of M37 lipase, despite the considerable sequence similarities. Meanwhile, MA1-3 lipase performed a transesterification reaction using olive oil and various alcohols including methanol, ethanol, 1-propanol, and 1-butanol. In the presence of t-butanol as a co-solvent, this lipase produced biodiesel using methanol and plant or waste oils. The highest biodiesel conversion yield (73%) was achieved using waste soybean oil and methanol at a molar ratio of 1:5 after 12 h using 5 units of lipase.

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs that function as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Yeast reductase YDL124W has been shown to convert ethyl 4-chloro-3-oxo butanoate (ECOB) into (S)-ECHB in an enantioselective manner. In that reduction, YDL124W absolutely prefers NADPH as a cofactor to NADH. NADPH is, however, much more expensive and unstable than NADH. In this study, four amino acid residues that directly interact with the 2′-phosphate group of adenosine ribose of NADPH were identified based on the homology model and docking model of YDL124W with ECOB and NADPH. Among various single mutants altered in these target residues, R264H evidenced noticeably increased reductase activity with NADH. The double mutant, R264H/R27Y, had more profoundly enhanced reductase activity with NADH. Kinetic analysis results demonstrated that these mutants had reduced Km values toward NADH and increased kcat/Km values in comparison with the wild-type. Computer modeling showed that no hydrogen bond could be formed between the wild-type and 2′-hydroxyl group of adenosine ribose of the NADH cofactor, whereas three hydrogen bonds and one π–π stacking interaction could be formed between R264H or R264H/R27Y mutants and the NADH cofactor. These two mutants could efficiently produce (S)-ECHB using NADH as a cofactor in conjunction with formate dehydrogenase as a coupling enzyme.

Lysine decarboxylase (CadA) converts

Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (

Two staphylococcal lipases were obtained from Staphylococcus epidermidis S2 and Staphylococcus aureus S11 isolated from sebaceous areas on the skin of the human face. The molecular mass of both enzymes was estimated to be 45 kDa by SDS-PAGE. S2 lipase displayed its highest activity in the hydrolysis of olive oil at 32 degrees C and pH 8, whereas S11 lipase showed optimal activity at 31 degrees C and pH 8.5. The S2 lipase showed the property of cold-adaptation, with activation energy of 6.52 kcal/mol. In contrast, S11 lipase's activation energy, at 21 kcal/mol, was more characteristic of mesophilic lipases. S2 lipase was stable up to 45° C and within the pH range from 5 to 9, whereas S11 lipase was stable up to 50 degrees C and from pH 6 to 10. Both enzymes had high activity against tributyrin, waste soybean oil, and fish oil. Sequence analysis of the S2 lipase gene showed an open reading frame of 2,067 bp encoding a signal peptide (35 aa), a pro-peptide (267 aa), and a mature enzyme (386 aa); the S11 lipase gene, at 2,076 bp, also encoded a signal peptide (37 aa), pro-peptide (255 aa), and mature enzyme (399 aa). The two enzymes maintained amino acid sequence identity of 98-99% with other similar staphylococcal lipases. Their microbial origins and biochemical properties may make these staphylococcal lipases isolated from facial sebaceous skin suitable for use as catalysts in the cosmetic, medicinal, food, or detergent industries.

Biodiesel is methyl and ethyl esters of long-chain fatty acids produced from vegetable oils or animal fats. Lipase enzymes have occasionally been used for the production of this biofuel. Recently, biodiesel production using immobilized lipase has received increased attention. Through enhanced stability and reusability, immobilized lipase can contribute to the reduction of the costs inherent to biodiesel production. In this study, methanol-tolerant lipase M37 from Photobacterium lipolyticum was immobilized using the cross-linked enzyme aggregate (CLEA) method. Lipase M37 has a high lysine content (9.7%) in its protein sequence. Most lysine residues are located evenly over the surface of the protein, except for the lid structure region, which makes the CLEA preparation yield quite high (-93%). CLEA M37 evidences an optimal temperature of 30°C, and an optimal pH of 9-10. It was stable up to 50°C and in a pH range of 4.0-11.0. Both soluble M37 and CLEA M37 were stable in the presence of high concentrations of methanol, ethanol, 1-propanol, and n-butanol. That is, their activities were maintained at solvent concentrations above 10% (v/v). CLEA M37 could produce biodiesel from olive oil and alcohols such as methanol and ethanol. Additionally, CLEA M37 generated biodiesel via both 2-step methanol feeding procedures. Considering its physical stability and reusability, CLEA M37 may potentially be used as a catalyst in organic synthesis, including the biodiesel production reaction.

(S)-3-Chloro-1-phenyl-1-propanol ((S)-CPPO) is a useful chiral building block for the synthesis of anti-depressant drugs. The yeast reductase, YOL151W, evidences enantioselective reduction activity, converting 3-chloro-1-phenyl-1-propanone (3-CPP) into (S)-CPPO. Escherichia coli whole cells co-expressing YOL151W and Bacillus subtilis glucose dehydrogenase were employed for the synthesis of CPPO following permeabilization treatment. A reaction system employing these recombinant E. coli whole cells could convert 30 mM 3-CPP enantioselectively into (S)-CPPO. In an effort to enhance substrate solubility and to prevent substrate/product inhibition during the enzyme reaction process, a variety of ionic liquids were tested and [Bmim][NTf2] was ultimately selected for the ionic liquid/water two phase system. Tween 40 was added to accomplish the efficient mixing of the two phases. Using these recombinant E. coli whole cells and the [Bmim][NTf2]/water two phase system, 100 mM (S)-CPPO was generated with an enantiomeric excess of >99%.

Rhodococcus erythropolis amidase was expressed in Escherichia coli cells. The crude amidase in the cell-free extract was immobilized using the cross-linked enzyme aggregate (CLEA) method. The crude amidase was mixed with bovine serum albumin and then precipitated with ammonium sulfate. The resultant precipitant was subsequently cross-linked with glutaraldehyde. Scanning electron microscopy revealed that this co-CLEA had a ball-like shape of a diameter of approximately 1 micronm. This co-CLEA evidenced hydrolytic activity toward a variety of amide substrates. The amidase co-CLEA evidenced an optimum temperature of 60 degrees and an optimum pH of 8.0, results that were similar to those of the soluble amidase. The reaction stability of the co-CLEA was increased. That is, it was stable up to 50 degrees and in a pH range of 5.0-12.0. Additionally, the co-CLEA could be recovered by centrifugation, and retained 96% activity after 3 repeated cycles. This amidase co-CLEA may prove useful as a substitute for soluble amidase as a biocatalyst in the pharmaceutical and chemical industries.

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40 degrees C and 7.5-8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0-7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.

The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α 2 β 2 . The purified enzyme had a high specific activity of 860 U mg −1 toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site. The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min −1 mg −1 . This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl ( S )-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin.

The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 A resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures. Proteins 2008. © 2008 Wiley-Liss, Inc.

Enantiomerically pure 2-chloromandelic acid esters are important chiral building blocks for synthesis of a wide range of pharmaceutical products, such as an anti-thrombotic agent, (S)-clopidogrel. An efficient and novel process for resolution of methyl 2-chloromandelate was developed by using a lipase-mediated transesterification. Among 11 hydrolytic enzymes examined, Candida antarctica lipase A (CAL-A) showed the highest enantioselectivity and reaction rate toward methyl (S)-2-chloromandelate. Methyl (R)-2-chloromandelate was obtained in enantiomerically pure form (>99% ee) and 41% yield through the lipase-mediated resolution under a solvent-free condition. CAL-A maintained its catalytic activity during 13 cycles of repeated use without significant decrease in enantioselectivity, indicating that the method is economical and easy to scale-up for commercial production of methyl (R)-2-chloromandelate.

A metagenomics approach is an efficient method of isolating novel and useful genes from uncultured microorganisms in diverse environments. In this research, a gene encoding a new esterase (EM2L8) was cloned and characterized from the metagenomic DNA library of a deep-sea sediment. The gene consisted of 804bp encoding a polypeptide of 267 amino acids with a molecular mass of 28,952. The deduced amino acid sequence showed similarities with the BioH of Kurthia, the 3-oxoadipate enol-lactonase of Haloarcula and the acyltransferase of Thermoanaerobacter, which feature identities of 38%, 32%, and 33%, respectively. Residues essential for esterase activity, such as pentapeptide (GXSXG) and catalytic triad sequences, were uncovered. While the protein was overproduced mainly as inclusion body at 37 degrees C, it was mainly produced as a soluble active enzyme at 18 degrees C. A zymogram analysis revealed that purified EM2L8 taken from the soluble fraction could hydrolyze tributyrin substrate. Furthermore, the protein from the inclusion body fraction also showed strong activity on gel, thus indicating that the protein was refolded during SDS-gel electrophoresis and the ensuing incubation period. When the inclusion body was mixed with some anionic detergent solutions and diluted with a non-detergent buffer, the insoluble EM2L8 refolded rapidly and recovered its full esterase activity. Although EM2L8 had an optimum temperature of 50-55 degrees C, its activation energy in the range of 10-40 degrees C was 8.34kcal/mol, indicating that it is a cold-adapted enzyme. Moreover, it was found to have an optimum pH of 10-11, thus revealing that it is an alkaline enzyme. In this paper, the new esterase EM2L8 buried in a deep-sea sediment became known on the surface and was characterized biochemically.

An organic solvent-stable lipase from Proteus vulgaris K80 was used to produce the omega-3 polyunsaturated fatty acid ethyl esters (ω-3 PUFA EEs). First, the lyophilized recombinant lipase K80 (LyoK80) was used to perform the transesterification reaction of menhaden oil and ethanol. LyoK80 produced the ω-3 PUFA EEs with a conversion yield of 82 % in the presence of 20 % water content via a three-step ethanol-feeding process; however, in a non-aqueous condition, LyoK80 produced only a slight amount of the ω-3 PUFA EEs. To enhance its reaction properties, the lipase K80 was immobilized on a hydrophobic bead to derive ImmK80; the biochemical properties and substrate specificity of ImmK80 are similar to those of LyoK80. ImmK80 was then used to produce ω-3 PUFA EEs in accordance with the same transesterification reaction. Unlike LyoK80, ImmK80 achieved a high ω-3 PUFA EE conversion yield of 86 % under a non-aqueous system via a one-step ethanol-feeding reaction. The ω-3 PUFA EEs were purified up to 92 % using a urea complexation method.

A sequence-based approach was used to retrieve functional lipases from microbial genome databases. Many novel genes assigned as putative lipases were tested using the criteria of the typical lipase sequence rule, based on a consensus sequence of a catalytic triad (Ser, Asp, His) and oxyanion hole sequence (HG). To obtain the lipase genes satisfying the sequence rule, PCR cloning was performed, while the lipase activities were tested using a tributyrin/tricaprylin plate and p-nitrophenyl caproate. Among nine putative lipases from four strains, five functional lipolytic proteins were obtained from Archaeoglobus fulgidus, Deinococcus radiodurans, and Agrobacterium tumefaciens. All five lipases exhibited a relatively low sequence similarity (less than 26.7%) with known lipases and turned out to belong to different lipase families. Accordingly, the current results indicate that the proposed strategic approach based on the microbial genome is an efficient and rapid method for finding novel and functional lipases.

A Bacillus niacini strain (EM001) producing an ofloxacin ester-enantioselective esterase was isolated from the soil samples collected near Taejon, Korea. The cloned gene showed that the esterase EM001 composed of 495 amino acids corresponding to a relative molecular weight (Mr) of 54,098 kDa. Based on the Mr and the protein sequence, the esterase EM001 was similar to p-nitrobenzyl esterase from Bacillus subtilis with an identity of 41.8%. The optimum temperature and pH of the purified His-tagged enzyme were 45 °C and 9.0, respectively. The purified esterase EM001 hydrolyzed preferably (R)-ofloxacin propyl ester than (S)-form ester at the initial reaction phase with an eeP of 67% until the conversion rate become up to 35%.

An esterase-producing Bacillus megaterium strain (20-1) was isolated from a soil sample collected in South Korea. The cloned gene showed that the esterase 20-1 composed of 310 amino acids corresponding to a molecular mass (Mr) of 34,638. Based on the Mr and the protein sequence, the esterase 20-1 belonged to the H lipase/esterase group. The optimum temperature and pH of the purified His-tagged enzyme were 20–35 °C and 8.0, respectively. The esterase 20-1 showed a ‘nonionic detergent-induced activation’ phenomenon, which was a detergent type- and concentration-dependent process. In comparison with the native enzyme, the Tween 80-treated enzyme had relatively a similar kcat value of 274 s−1 but a very low Km value of 0.037 mM for PNPC (C6), therefore, it showed a 14-fold increase in kcat/Km value.

Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was 60°C, which was 20°C higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to 40°C and in a pH range of 5.0- 10.0, whereas soluble K80 maintained its activity up to 35°C and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.

An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET-22b(+) expression vector and its expression was induced by IPTG at 30 degrees C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26% of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62%. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68 degrees C and it was stable up to 55 degrees C for 30 min-incubation. The thermostability increased by about 8-10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66 degrees C in the presence of calcium ions but at 58 degrees C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures.