A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Purification and characterization of a novel solvent-tolerant lipase from Fusarium heterosporum

A microorganism producing a solvent-tolerant lipase was identified as Fusarium (F.) heterosporum. The lipase was purified from the culture filtrate to homogeneity as judged by disc-PAGE and SDS-PAGE. The purification included SP-Sephadex chromatography, gel filtration and isoelectric focusing, and the recovery yield was 38%. The lipase was a monomeric protein with a molecular weight of 31 kDa estimated by SDS-PAGE, and a pI of 7.0. The optimum pH at 40°C and optimum temperature at pH 5.6 were 5.5–6.0 and 45–50°C, respectively, when olive oil was used as the substrate. The lipase was stable over a pH range of 4–10 at 30°C for 4 h, and up to 40°C at pH 5.6 for 30 min. Furthermore, the enzyme was not inactivated even after incubation at 30°C in 50% solvent such as dimethylsulfoxide (DMSO), hexane, benzene and ether for 20 h. The activity did not decrease in a reaction with stirring in a mixture containing 50% DMSO or dimethylformamide. The lipase preferably reacted on middle-chain fatty acid triglycerides (6≤C≤12), and cleaved only 1,3-ester bonds of triolein. The enzyme had an N-terminal sequence of Ala-Val-Thr-Val-Thr-Thr-Gln-Asp-Leu-Ser, which has not previously been found in any other protein. We compared the properties of lipases from F. heterosporum and another strain F. oxysporum.     

Production and activity of extracellular lipase from Luteibacter sp.

Microbial lipases are widely used in industrial applications due to their versatility, and the characterization of new lipase-producing microorganisms could provide new sources of these enzymes, with different specificities and better activities. In this context, we have improved lipase production by Luteibacter sp. by using basal medium supplemented with 2 % olive oil, a pH of 6 and a growth temperature of 37 °C. The enzyme extraction process with the addition of 0.25 % Tween 80 increased lipase activity. Implementation of these modifications increased lipase activity by approximately 430 %. The lipase activities produced in the culture supernatant (LCS) and extracted with Tween 80 (LCST80) were characterized. Both extracts hydrolyzed ρ-nitrophenyl (ρNP) esters with different acyl chain lengths, with a preference for short acyl lengths, and had optimum activity at 45 °C. The LCS was stable at acidic and alkaline pH, but LCST80 was only stable at alkaline pH. Methanol, SDS, Triton X-100, EDTA, and EGTA did not affect lipase activity, while divalent cations (Ca²⁺, Zn²⁺, Mg²⁺) - with the exception of Co²⁺— increased lipase activity. Both extracts showed transesterification activity on ρNP ester substrates, and both were able to hydrolyze different natural lipids. The characterization of lipase produced by Luteibacter sp. introduces this recently described genus as a new source of lipases with great biotechnological potential.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.     

An Arginine Specific Protease from Spirulina platensis

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (K_m = 5.5 × 10⁻⁶ M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (K_i = 1.1 × 10⁻⁸ M) and leupeptin (K_i = 3.9 × 10⁻⁸ M) but was not inhibited with N^α-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.     

Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis

Strains of thermophilic bacilli were screened for cellulolytic activity by gel diffusion assay on selective medium at 55°C. Strain B-41361, identified as a strain of Bacillus licheniformis, displayed activity against carboxymethylcellulose. Zymogram analysis demonstrated several catalytically active polypeptides with the most prominent species having a mass of 37 kDa. The enzyme was purified 60-fold with a 17% yield and specific activity of 183 U/mg. The amino terminal sequence was homologous to members of glycoside hydrolase family 5. Optimal temperature was 65°C (measured over 30 min), but the enzyme was most stable at 60°C, retaining greater than 90% activity after one hour. The enzyme had a broad pH range, with maximal activity at pH 6.0, 75% maximal activity at pH 4.5, and 40% at pH 10. The enzyme hydrolyzed p-nitrophenylcellobioside, barley β-glucan, and lichenan, but no activity was detected against avicel or acid-swollen cellulose.Mention of a trade name or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Screening Botryosphaeria species for lipases: Production of lipase by Botryosphaeria ribis EC-01 grown on soybean oil and other carbon sources

Nine isolates of Botryosphaeria spp. were screened for lipases when cultivated on eight different plant seed oils and glycerol, and all produced lipases. Botryosphaeria ribis EC-01 produced highest lipase titres on soybean oil and glycerol, while eight isolates of Botryosphaeria rhodina produced significantly lower enzyme titres. B. ribis EC-01 produced lipase when grown on different fatty acids, surfactants, carbohydrates and triacylglycerols, with highest enzyme titres produced on Triton X-100-emulsified stearic (316.7 U/mL), palmitic (283.5 U/mL) and oleic (247.4 U/mg) acids, and soybean oil (105.6 U/mL), as well as castor oil (191.2 U/mg); an enhancement of 9-fold over soybean oil-grown cultures. Glycerol was also a good substrate for lipase production. The crude lipase extract was optimally active at pH 8.0 and 55 °C, stable between 30 and 55 °C and pH 1–10, and tolerant to 50% (v/v) glycerol, methanol and ethanol. The crude lipase showed affinity for substrates of short, average and long-chain fatty acids (different esters of p-nitrophenol and triacylglycerols). Zymograms developed with 4-methylumbelliferyl-butyrate showed two bands of lipolytic activity at 45 and 15 kDa. This is the first report on the production of lipases by B. ribis grown on these different carbon sources.     

Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95

GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80 % lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.     

Purification of extracellular lipases from Trichosporon fermentans WU-C12

Two types of extracellular lipases (I and II) from Trichosporon fermentans WU-C12 were purified by acetone precipitation and successive chromatographies on Butyl-Toyopearl 650 M, Toyopearl HW-55F and Q-Sepharose FF. The molecular weight of lipase I was 53 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and 160 kDa by gel filtration, while that of lipase II was 55 kDa by SDS-PAGE and 60 kDa by gel filtration. For the hydrolysis of olive oil, the optimum pH and temperature of both the lipases were 5.5 and 35°C, respectively. The lipases showed stable activities after incubation at 30°C for 24 h in a pH range from 4.0 to 8.0. The thermostability of lipase I for 30 min at a reaction pH of 5.5 was up to 40°C, while that of lipase II under the same conditions was up to 50°C. Both lipases could hydrolyze the 1-, 2-, and 3-positions of triolein, and cleave all three ester bonds, regardless of the position in the triglyceride.     

Characterization of a Novel Alkalophilic Lipase From <Emphasis Type="Italic">Aneurinibacillus thermoaerophilus</Emphasis>: Lid Heterogeneity and Assignment to Family I.5

Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase α/β hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding. The enzyme was active from 25 to 65 °C under neutral pH, but its maximal activity was detected at pH 10 and 45 °C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 °C and pH 10 followed by cooling prevented the restoration of activity, while similar treatments performed at pH 8 (where folded aggregates may form) allowed recovery of 50% of the initial activity. In silico analyses revealed a high conservation (85% or more) for the main lipase signature sequences in LipAT despite an overall low residue identity (60% identity compared to family I.5 lipases). In contrast, the active site lid region in LipAT is very distinct showing only 25% amino acid sequence identity to other homologous lipases in this region. Comparison of lids among lipases from the I.5 family members and LipAT reveals that this region should be a primary target for elucidation, optimisation and prediction of structure–function relationships in lipases.     

Lipase from Candida paralipolytica

The lipase from Candida paralipolytica was purified, as judged by disc electrophoresis. The purification was about 132 fold, based on protein, with a recovery of 32% from the acetone precipitate of the cultivated broth.

After purification, modification of the enzyme was performed by dialyzing its solution against 1 m sodium chloride in acetate buffer at room temperature and by separating the modified enzyme from an unknown substance(s) with a Sephadex G–75 column.

The optimum pH for lipolysis of the purified lipase was 8.0, while that of the modified one was 7.0. Sodium taurocholate was required essentially by the purified enzyme, but not by the modified one. The purified lipase was stable below 37°C and in the pH range from 3.5 to 9.0 at 5°C.     

Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634

Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K _m) and the maximum reaction velocity (V _max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities.     

Biochemical and Structural Characterization of Two Site-Directed Mutants of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> Lipase

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all enzymes retained their initial activities.     

Expression and characterization of a novel heterologous moderately thermostable lipase derived from metagenomics in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Seven lipolytic genes were isolated and sequenced from a metagenomic library that was constructed following biomass enrichment in a fed-batch bioreactor submitted to high temperature (50–70°C) and alkaline pH (7–8.5). Among those sequences, lipIAF1-6 was chosen for further study and cloned in Streptomyces lividans 10–164. The G+C content within the sequence was 64.3%. The encoded protein, LipIAF1-6, was related to various putative lipases previously identified in different genome sequences. Homology of LipIAF-6 with the different lipases did not exceed 31%. The optimum pH (8.5) and temperature (60°C) of the purified enzyme were in agreement with the enrichment conditions. Furthermore, the enzyme was thermostable for as long as 30 min at 70°C. The maximum activity of the purified lipase was 4,287 IU/mg towards p-nitrophenyl (p-NP) butyrate (60°C; pH 8.5). LipIAF1-6 does not seem to need the presence of metal ions for its activity. The enzyme was slightly inhibited by 10 mM CoCl₂ (14%), HgCl₂ (12%), and dithiothreitol (DTT) (15%). The serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF) reduced activity by 39% and 71% when incubated at concentrations of 1 and 10 mM, respectively. Finally, LipIAF1-6 was stable in different organic solvents, and against several surfactants and oxidative agents commonly found in detergent formulations. These results are quite encouraging for further use of this enzyme in different industrial processes.     

Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.     

Screening of filamentous fungi for lipase production: Hypocrea pseudokoningii a new producer with a high biotechnological potential

Abstract

Filamentous fungi isolated from soil samples were screened for extracellular lipase production. The best producer was Hypocrea pseudokoningii identified by taxonomical criteria, and by rDNA sequencing of the variable internal transcribed spacers (ITS I and II) and the intervening 5.8S gene. The fungus was grown in a complex medium supplemented with 1% Tween 80 and 0.2% yeast extract, for 4 days. The optimum pH for extracellular and intracellular lipases was 7.0 and 8.0, respectively. Both enzymes exhibited maximum activity at 40°C. Extracellular and intracellular lipase activities were highly stable in the pH range 3.0–8.0 at room temperature. The intracellular lipase was thermostable up to 60°C, for 15 min and the extracellular, for 107 min, at the same temperature. The intracellular lipase was stimulated by silver ions. Extracellular lipase was stable in organic solvents, such as DMSO, alcohols, acetone, and acetonitrile, for 24 hours. Lipase activity increased around 80% when detergents were added to the enzymatic assay, such as Tween 80, Triton X-100, and SDS.     

Extracellular solvent stable cold-active lipase from psychrotrophic Bacillus sphaericus MTCC 7526: partial purification and characterization

Psychrotropic Bacillus sphaericus producing solvent stable cold-active lipase upon growth at low temperature was isolated from Gangotri glacier. Optimal parameters for lipase production were investigated and the strain was able to produce lipase even at 15 °C. An incubation period of 48 h and pH 8 was found to be conducive for cold-active lipase production. The addition of trybutyrin as substrate and lactose as additional carbon source increased lipase production. The enzyme was purified up to 17.74-fold by ammonium sulphate precipitation followed by DEAE cellulose column chromatography. The optimum temperature and pH for lipase activity were found to be 15 °C and 8.0, respectively. The lipase was found to be stable in the temperature range 20–30 °C and the pH range 6.0–9.0. The protein retained more than 83 % of its initial activity after exposure to organic solvents. The lipase exhibited significant stability in presence of acetone and DMSO retaining >90 % activity. The enzyme activity was inhibited by 10 mM CuSO₄ and EDTA but showed no loss in activity after incubation with other metals or inhibitors examined in this study.     

Isolation of the Volatile Components of Fresh Onion by Thermal Desorption Cold Trap Capillary Gas Chromatography

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45°C and the half-life was 1 h at 64°C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chainfatty acid esters. The enzyme had a K_M of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37°C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Lepidopterous Sex Attractants

Fusobacterium K-60, a ginsenoside R_b1-metabolizing bacterium, was isolated from human intestinal feces. From this Fusodobacterium K-60, a ginsenoside R_b1-metabolizing enzyme, β-glucosidase, has been purified. The enzyme was purified to apparent homogeneity by a combination of butyl-Toyopearl, hydroxyapatite ultragel, Q-Sepharose, and Sephacryl S-300 HR column chromatographies with a final specific activity of 1.52 μmol/min/mg. It had optimal activity at pH 7.0 and 40°C. The molecular mass of this purified enzyme was 320 kDa, with 4 identical subunits (80 kDa). The purified enzyme activity was inhibited by Ba⁺⁺, Fe⁺⁺, and some agents that modify cysteine residues. This enzyme strongly hydrolyzed sophorose, followed by p-nitrophenyl β-D-glucopyranoside, esculin, and ginsenoside R_b1. However, this enzyme did not change 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901) to 20(S)-protopanaxadiol, while it weakly changed ginsenoside R_b1 to IH-901. These findings suggest that the Fusobacterial β-glucosidase is a novel enzyme transforming ginsenoside R_b1.     

Properties of a novel β-glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2

A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60 °C, and the deactivation energy (E_d) for FPG was 88.6 kJ mo1⁻¹. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg₃. The enzyme hydrolyzed the 3-C, β-(1 → 2)-glucoside of ginsenoside Rg₃ to produce ginsenoside Rh₂, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh₂. The K_m and V_max values of FPG for ginsenoside Rg₃ were 2.37 mM and 0.568 μmol (h mg protein)⁻¹, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides.