A Cold-Adapted and Organic Solvent-Tolerant Lipase from a Psychrotrophic Bacterium Pseudomonas sp. Strain YY31: Identification, Cloning, and Characterization

A novel cold-adapted lipase (designated as LipYY31) was obtained from a psychrotrophic Pseudomonas sp. YY31. The strain YY31 was gram-negative, rod shaped, motile by means of one polar flagellum, and exhibited chemotaxis toward oil droplets under a microscope. The strain displayed remarkable degradation of edible oil and fat even at 5 °C. The LipYY31 DNA fragment contains an open reading frame of 1,410 bp which encoded a protein of 470 amino acids with an estimated molecular mass of 49,584 Da. LipYY31 showed high sequence similarity to those of subfamily Ι.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 25–30 °C, and it retained 20–40 % of its activity at 0–5 °C. The optimal pH value was 8.0. The activity was strongly inhibited by Cd²⁺, Zn²⁺, EDTA and was highly dependent on Ca²⁺. Tricaprin and p-nitrophenyl caprate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipYY31 also had high activity towards natural substrates including edible vegetable oils and animal fat. Furthermore, LipYY31 was very active and stable in the presence of several detergents and organic solvents. In particular, the lipase exhibited high stability against organic solvents such as methanol, ethanol, and isopropanol.     

Expression and Characterization of a Novel Enantioselective Lipase from Aspergillus fumigatus

A 1,080-bp cDNA (CGMCC 2873) encoding of a cold-active lipase of Aspergillus fumigatus (AFL67) was cloned and expressed in Escherichia coli for the first time. The new lipase, AFL67, was one-step purified by 8.30 folds through Ni?CNTA affinity chromatography with a recovery of 86.8?%. The specific activity of purified AFL67 was 449?U?mg^?1 on p-NP hexanoate. AFL67 preferentially hydrolyzed p-nitrophenyl esters of short- and medium-chain fatty acids, with p-nitrophenyl hexanoate the maximum. The optimum temperature and pH was 15?°C and 7.5, respectively. The purified AFL67 was stable at 10?C25?°C for 30?min, and in the pH range of 6.0?C9.0 for 16?h (at 4?°C). Its activity was increased by 47 and 50?%, in the presence of 10?% (v/v) ethanol and isopropanol, respectively. The new lipase AFL67 highly enantioselectively deacylated (S)-??-acetoxyphenylacetic acid (APA) and o-Cl-APA, m-Cl-APA, and p-Cl-APA to (S)-mandelic acid and its derivates. These features render this cold-active novel lipase AFL67 attractive for biotechnological applications in the field of enantioselective synthesis of chiral mandelic acids, o-acylated mandelic acids, and their derivates and detergent additives.     

Transesterification of Waste Cooking Oil by an Organic Solvent-Tolerant Alkaline Lipase from Streptomyces sp. CS273

The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0?～?9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo₄, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis.     

Purification and Characterization of an Organic Solvent-Tolerant Lipase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> CS-2

An extracellular lipase secreted by Pseudomonas aeruginosa CS-2 was purified to homogeneity about 25.5-fold with an overall yield of 45.5%. The molecular mass of the lipase was estimated to be 33.9 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum temperature and pH were 50 °C and 8.0. The lipase was found to be stable at pH 4–10 and below 50 °C. Its hydrolytic activity was highest against p-nitrophenyl palmitate (p-NPP) among p-nitrophenyl esters of fatty acids with various chain lengths. The lipase was activated in the presence of Ca²⁺, while it was inactivated by other metal ions more or less. EDTA significantly reduced the lipase activity, indicating the lipase was a metalloenzyme. Gum Arabic and polyvinyl alcohol 124 enhanced lipase activity but Tween-20, Tween-80, and hexadecyltrimethyl ammonium bromide strongly inhibited the lipase. It exhibited stability in some organic solvents. The lipase was activated in the presence of acetonitrile. Conversely, it was drastically inactivated by methanol and ethanol.     

Screening, Gene Sequencing and Characterising of Lipase for Methanolysis of Crude Palm Oil

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/β hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K _M of 0.75 mM and a V _max of 0.33 mM?min^?1 on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.     

Protein-Coated Microcrystals of Pseudomonas aeruginosa PseA lipase

Highly active Pseudomonas aeruginosa lipase protein-coated microcrystals (PAL PCMC) have been prepared by immobilization of protein onto K₂SO₄ as excipient solid support carrier and n-propanol as precipitating solvent. Transmission electron micrographs confirmed the formation of PAL PCMC. These PCMC were found to be a catalytically more active and stable preparation for p-nitrophenyl palmitate hydrolysis in n-heptane, compared to free lipase. The V _max, K _m, and temperature optimum for PAL PCMC increased from 0.49 to 5.66 nmol min^?1 mg^?1, 589 to 679.8 mmol, and 40°C to 45°C, respectively. These were thermally more stable with 4.65, 2.56, and 1.24-fold improvement in half lives at 45°C, 55°C, and 60°C compared to free P. aeruginosa PseA lipase. Their catalytic efficiency was enhanced by tenfold over that of free enzyme. PAL PCMC offer a simple and effective technique for obtaining stable and efficient lipase preparation for biocatalysis in nonaqueous medium.     

The Purification and Characterization of Lipases from <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis>, and Their Immobilization and Use for Biodiesel Production from Coconut Oil

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)—purified 25.41-fold, recovery of 47.1%—and lipase B (32,000 Da)—purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca²⁺, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5–10.0 and 20–80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.     

Purification and characterization of an extracellular lipolytic enzyme from the fermented fish-originated halotolerant bacterium, <Emphasis Type="Italic">Virgibacillus alimentarius</Emphasis> LBU20907

A halotolerant Virgibacillus alimentarius LBU20907 isolated from fermented fish (Budu) was found to be an efficient producer of extracellular halophilic lipase enzyme. The enzyme was purified 5.99-fold with a 0.15% final yield to homogeneity by ammonium sulfate precipitation, followed by dialysis, Toyopearl DEAE-650 M ion exchange chromatography, Toyopearl butyl-650 M hydrophobic interaction chromatography, and Toyopearl-HW 55 F gel filtration chromatography. SDS-PAGE of purified lipase exhibited a homogenous single band with a very high molecular weight of 100 kDa. The properties of purified lipase revealed maximum activity at pH 7.0 and 40 °C. It was also highly stable in a pH range of 6.0–7.0, retaining more than 90% activity for 24 h. It was stable at the temperature of 30–50 °C and maintained more than 80% activity for 16 h. The purified lipase performing the maximal activity in the presence of 20.0% NaCl indicated halophilic enzyme properties. Its lipolytic activity was highest against p-nitrophenyl palmitate. The lipase activity was found to be enhanced in hexane. The enzyme activity was stimulated in the presence of Zn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺; while, it was completely inhibited by Ba²⁺ and Co²⁺. The enzyme had a K _m and V _max of 108.0 mg and 79.1 U mL^?1, respectively.     

Role of Met93 and Thr96 in the Lid Hinge Region of Rhizopus chinensis Lipase

We engineered Rhizopus chinensis lipase to study its critical amino acid role in catalytic properties. Based on the amino acid sequence and three-dimensional model of the lipase, residues located in its lid hinge region (Met93 and Thr96) were replaced with corresponding amino acid residues (Ile93 and Asn96) found in the lid hinge region of Rhizopus oryzae lipase. The substitutions in the lid hinge region affected not only substrate specificity but also the thermostability of the lipase. Both lipases preferred p-nitrophenyl laurate and glyceryl trilaurate (C12). However, the variant S4-3O showed a slight decline in activity toward long-chain fatty acid (C16–C18). When enzymes activities decreased by half, the temperature of the variant (45 °C) was 22 °C lower than the parent (67 °C), probably substantially destabilized the structure of the lid region. The interfacial kinetic analysis of S4-3O suggested that the lower catalytic efficiency was due to a higher K _m* value. According to the lipase structure investigated, Ile93Met played a role of narrowing the size of the hydrophobic patch, which affected the substrate binding affinity, and Asn96Thr destabilized the structure of the lipase by disrupting the H-bond interaction in the lid region.     

Production of Thermostable Lipase by Thermomyces lanuginosus on Solid-State Fermentation: Selective Hydrolysis of Sardine Oil

A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus.     

Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii

A gene coding for the extracellular esterase (EstK) was cloned from the psychrotrophic bacterium Pseudomonas mandelii based on its partial amino acid sequence as determined by mass spectrometry. The entire open reading frame consisting of 1,011 bp was expressed in Escherichia coli as a soluble protein and purified by nickel-chelated affinity chromatography and Capto Q column chromatography. Here, we show that the 33-kDa recombinant EstK protein (rEstKsp) had a substrate preference for esters of short-chain fatty acids, especially, p-nitrophenyl acetate. Optimum activity of rEstKsp was at pH 8.5 and 40 °C. The esterase activity remained similar from a range of 4～20 °C, but the maximum activity varied depending upon pH. With p-nitrophenyl acetate as the substrate, K _M was 210 μM and k _cat was 3.4 s^?1. Circular dichroism and fluorescence spectroscopy results revealed that rEstKsp had a predominantly α-helical structure and maintained its folded state at 4～40 °C. Interestingly, the tertiary structure of rEstKsp was predicted based on the structures of other hyperthermophilic esterases. Our results demonstrated that both native and rEstKsp are active at low temperatures and have a unique substrate preference for p-nitrophenyl acetate.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Significantly Improved Expression and Biochemical Properties of Recombinant Serratia marcescens Lipase as Robust Biocatalyst for Kinetic Resolution of Chiral Ester

A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 × 10⁵ U/l, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 °C and within the pH range of 5.0−6.8, giving temperature and pH optima at 40 °C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca²⁺, Cu²⁺, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C₁₂). Using racemic trans-3-(4′-methoxy-phenyl)-glycidyl methyl ester [(±)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene–water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 °C under shaking condition (160 rpm), affording (−)-MPGM in nearly 100% ee. The K _m and V _max values of the lipase for (±)-MPGM were 222 mM and 1.24 mmol min⁻¹ mg⁻¹, respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.     

Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases

Environmental DNA from soil and water samples was extracted to construct a plasmid library and a fosmid library containing 19,500 and 20,400 clones, respectively. Two esterases (EstP2K and EstF4K) were finally isolated from each library based on activity screening, and both of them were characterized in this study. The esterase EstF4K consists of 396 amino acids with an SMTK motif which belongs to family VIII esterase/lipase. The amino acid sequence of EstF4K showed 83 % identity with that of EstA3, a reported esterase isolated from uncultured organisms of soil. EstP2K is composed of 224 amino acids in size and shows only 37 % identity with a putative lipase of Neisseria elongata subsp. The purified EstF4K was optimally active at pH?8.0 and 50 °C. It was remarkably active and very stable in the presence of 30 % dimethyl sulfoxide. Activity fingerprint of EstF4K displayed a higher level of activity toward short-chain fatty acid p-nitrophenyl (pNP) esters, while EstP2K preferred bias for pNP caprylate ester. The optimum reaction temperature and pH for EstP2K are 45 °C and 7.5, respectively, and the enzyme exhibited strong tolerance in the presence of 30 % methanol. EstF4K and EstP2K showed opposite enantioselectivity for methyl 3-phenylglycidate, a chiral synthon for the synthesis of Taxol® side chain.     

Purification and Biochemical Characterization of a Novel Alkaline (Phospho)lipase from a Newly Isolated Fusarium solani Strain

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH₂-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca²⁺ and bile salts. The specific activities of FSL were about 1,610 and 2,414 U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH 8.0 and 37 °C. The (phospho)lipase enzyme was stable in the pH range of 5–10 and at temperatures below 45 °C.     

Sugar Ester Synthesis by Thermostable Lipase from <Emphasis Type="Italic">Streptomyces thermocarboxydus</Emphasis> ME168

The extracellular lipase from Streptomyces thermocarboxydus ME168 was purified to 9.5-fold with 20% yield, following concentration by acetone precipitation, ion exchange chromatography (Resource Q) and gel filtration chromatography (Superdex 200), respectively. The purified enzyme had an apparent molecular mass of 21 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of the lipase was ASDFDDQILG and was different from most other reported lipase. The enzyme showed maximum activity at 50 °C with the half-life of 180 min at 65 °C. It showed high stability at a broad pH range of 5.5–9.5 and was thermostable at the temperature range of 25–60 °C. The K _m and V _max were 0.28 mM and 1,428 U/mg, respectively, using p-nitrophenyl palmitate as substrate. It was active toward p-nitrophenyl ester with medium to long acyl chain (C₈–C₁₆). Lipase activity was inhibited by Zn²⁺, dithiothreitol (DTT), EDTA and some organic solvents, e.g., ethanol, acetone, dioxane, acetronitrile, tert-butanol and pyridine. Immobilized crude lipase of S. thermocarboxydus ME168 on celite could be used to synthesize sugar esters from glucose and vinyl acetate, vinyl butyrate or vinyl caproate in tert-butanol:pyridine (55:45 v/v) at 45 °C with conversion yields of 93, 67 and 55%, respectively.     

Molecular Cloning and Expression of a Novel β-Glucosidase Gene from Phialophora sp. G5

A novel β-glucosidase gene, bgl1G5, was cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the gene consists of a 1,431-bp open reading frame encoding a protein of 476 amino acids. The deduced amino acid sequence of bgl1G5 showed a high identity of 85 % with a characterized β-glucosidase from Humicola grisea of glycoside hydrolase family 1. Compared with other fungal counterparts, Bgl1G5 showed similar optimal activity at pH 6.0 and 50 °C and was stable at pH 5.0–9.0. Moreover, Bgl1G5 exhibited good thermostability at 50 °C (6 h half-life) and higher specific activity (54.9 U mg^–1). The K _m and V _max values towards p-nitrophenyl β-d-glucopyranoside (pNPG) were 0.33 mM and 103.1 μmol?min^–1?mg^–1, respectively. The substrate specificity assay showed that Bgl1G5 was highly active against pNPG, weak on p-nitrophenyl β-d-cellobioside (pNPC) and p-nitrophenyl-β-d-galactopyranoside (ONPG), and had no activity on cellobiose. This result indicated Bgl1G5 was a typical aryl β-glucosidase.     

Production, Purification, and Characterization of a β-Glucosidase of Penicillium funiculosum NCL1

Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of ??-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular ??-glucosidase production was studied in submerged fermentation. At 30?°C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of ??-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major ??-glucosidase (Bgl3A) with the apparent molecular weight of ~120?kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60?°C, and exhibited a half-life of 1?h at 60?°C. K _m of Bgl3A was found to be 0.057?mM with p-nitrophenyl ??-d-glucoside and V _max was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K _i of 1.5?mM. Bgl3A readily hydrolyzed glucosides with ??-linkage. Bgl3A activity was enhanced (156%) by Zn²⁺ and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.     

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH?7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40?°C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.     

Kinetic and stability characterization ofChromobacterium viscosum lipase and its comparison withPseudomonas glumae lipase

A kinetic study ofChromobacterium viscosum lipase was undertaken, and compared withPseudomonas glumae lipase. Optimum operation conditions were pH 9.0 and 50°C for both enzymes. A substrate specificity study was also developed. Both enzymes showed higher activity on triglycerides with a long chain of fatty acid; the specific activity was always higher for C.viscosum lipase. Stability of both enzymes in aqueous medium at 60°C and pH 9.0 was evaluated. C.viscosum lipase was three times more stable than P.glumae lipase, with at _1/2 value of 0.75 h. In addition, the activity of C.viscosum lipase with substrate concentration was studied with a triolein emulsion. A dependence of the intrinsic characteristics of the emulsion was observed. Therefore, stability ofC. viscosum lipase B with reaction products was assayed in a micellar system. Acid products reduced the specific activity of the enzyme. Glycerol and high buffer concentration were stabilizers of enzyme deactivation. Finally, substrate specificity ofC. viscosum lipase B in a micellar system was developed with tributyrin, tricaprylin, and triolein. Only tributyrin showed an apparent Michaelis-Menten kinetic with V_max ^app = 958 U/mg and K_ma ^app = 75.5 mM. Tricaprylin and triolein showed diffusion limitations at low substrate concentration and substrate inhibition at high substrate concentration. Diffusion parameters were calculated for both these substrates. Mass transfer coefficients (k₁) were 0.314 Å/min and 1.53 Å/min for tricaprylin and triolein, respectively. Effectiveness factors (η) were 0.536 and 0.768 for tricaprylin and triolein, respectively.