Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties

Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO₄.7H₂O, 0.05% KCl, 0.2% K₂HPO₄ and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme retained more than 90% of its activity. Enzyme activity was inhibited by Hg²⁺ and K⁺, whereas Ca²⁺ and Mn²⁺ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity.     

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.     

Immobilization in the presence of Triton X-100: modifications in activity and thermostability of <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> CCR11 lipase

A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.     

Production of Extracellular Acid Proteases by Aspergillus Clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 °C. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1%(w/v) at 30 °C at the third day of incubation. Cultures developed in Vogel medium with glucose at 2%(w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 30, 10 and 5 min, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physicochemical Properties of Niigata Waxy Rices

The crude lipase powder has been purified 216-fold in specific activity by means of pH adjustment, DEAE-Cellu1ose, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 column chromatography and the recovery of the activity was 30%. The purified lipase was confirmed to be homogeneous with disc electrophoresis and ultracentrifugal analyses. The purified lipase was stable in the pH range from 7.0 to 10.0. Optimal pH for the lipolysis of polyvinyl alcohol-emulsified olive oil at 45°C was 8.0 and optimal temperature was 60°C. The purified lipase was stable up to 60°C and retained 55% of full activity after heating at 70°C for 20 min.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

Purification and Properties of a Protease Produced by Bacillus Subtilis CN2 Isolated from a Vietnamese Fish Sauce

Bacillus subtilis CN2 isolated from a Vietnamese fish sauce produced a large quantity of an alkaline protease, when grown on a soy peptone medium. The protease was purified to an electrophoretically homogeneous state and crystallized in its pure condensed solution. The molecular weight was determined to be 27,636 Da, and the N-terminal amino acid sequence was AQSVPYGISQIKAPAL. The optimum pH and temperature were pH 10.0 and 50 °C, respectively. The protease was active over a wide pH range of pH 7.0–11.0, and also active over a broad temperature range of 30–60 °C. The enzyme was potently inhibited by 1 mM phenylmethanesulphonyl fluoride, but resistant to 1 mM sodium dodecyl sulphate (SDS). This revised version was published online in July 2006 with corrections to the Cover Date.     

Optimization of Culture Conditions for the Production of Extracellular Agarases from Newly Isolated Pseudomonas Aeruginosa AG LSL-11

Summary An agar-degrading bacterium capable of utilizing agar as sole source of carbon and energy was isolated from sea water by enrichment culture technique. The bacterium was identified as Pseudomonas aeruginosa and the culture conditions were standardized for the maximal production of extracellular agarases. The bacterium grew in the pH range 5.0–11.0, optimal between pH 7.0 and 8.0; temperature between 25 °C and 37 °C, optimal at 30 °C and sodium chloride concentration 0–8% and optimal at 2% respectively. The agarases secreted by Pseudomonas aeruginosa AG LSL-11 were inducible by agar and not by any other simple sugars tested. Maximal agarase production was observed at pH 8.0, and temperature 30 °C. The bacterium had no requirement for NaCl for both growth and production of agarases. The bacterium did not utilize other polysaccharides like ĸ-carrageenan, alginate, cellulose and CMC. The activity staining of partially purified agarase preparation after native-PAGE revealed the presence of three different agarases, agarase LSL-11a, LSL-11b and LSL-11c, whose molecular weights were estimated to be 76, 64 and 46 kDa respectively.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Lipase from marine strain using cooked sunflower oil waste: production optimization and application for hydrolysis and thermodynamic studies

The marine strain Pseudomonas otitidis was isolated to hydrolyze the cooked sunflower oil (CSO) followed by the production of lipase. The optimum culture conditions for the maximum lipase production were determined using Plackett–Burman design and response surface methodology. The maximum lipase production, 1,980 U/ml was achieved at the optimum culture conditions. After purification, an 8.4-fold purity of lipase with specific activity of 5,647 U/mg protein and molecular mass of 39 kDa was obtained. The purified lipase was stable at pH 5.0–9.0 and temperature 30–80 °C. Ca²⁺ and Triton X-100 showed stimulatory effect on the lipase activity. The purified lipase was highly stable in the non-polar solvents. The functional groups of the lipase were determined by Fourier transform-infrared (FT-IR) spectroscopy. The purified lipase showed higher hydrolytic activity towards CSO over the other cooked oil wastes. About 92.3 % of the CSO hydrolysis was observed by the lipase at the optimum time 3 h, pH 7.5 and temperature 35 °C. The hydrolysis of CSO obeyed pseudo first order rate kinetic model. The thermodynamic properties of the lipase hydrolysis were studied using the classical Van’t Hoff equation. The hydrolysis of CSO was confirmed by FT-IR studies.     

Production of a fibrinolytic enzyme by thermophilic Streptomyces species

A strong fibrin-specific fibrinolytic enzyme was purified from the cell-free spent culture broth of a thermophilic organism, Streptomyces megasporus SD5. The strain could produce 150 mg crude protein per litre of spent broth, with a specific activity of 80 IU (Plough units) per milligram, within 18 h of incubation at 55 °C in glucose yeast/extract/peptone (GYP) medium, pH 8.0. For production of the enzyme, the strain could utilize different carbon and nitrogen sources with a C:N ratio of ∼ 1:2. The enzyme was stable at a broad range of pH ranging from 5 to 9, and highly thermostable with 50% activity after storage at 60 °C for 6 months. The enzyme belonged to the serine endopeptidase group. In vitro clot lysis revealed that the enzyme was active at 37 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Production and partial characterization of alkaline feruloyl esterases by Fusarium oxysporum during submerged batch cultivation

Production of feruloyl esterases (FAEs) by Fusarium oxysporum was enhanced by optimization of initial pH of the culture medium, the type and concentration of nitrogen and carbon source. Submerged batch cultivation in a laboratory bioreactor (17 l) produced activity at 82 nkat g⁻¹ dry substrate (corn cobs) which compared favorably to those reported for the other microorganisms. Use of de-esterified corn cobs as carbon source decreased FAE production by 5.5-fold compared to untreated corn cobs even though ferulic acid (FA) was added to the concentration found in alkali-extracts of corn cobs. Production of FAE does not therefore, require FA, however, production is diminished by the removal of esterified FA from the growth substrate. Optimal FAE activity was observed at pH 7 and 50 °C with 68 and 55% activity at pH 8 and pH 9, respectively. The esterase was fully stable at pH 5–8 and up to 40 °C and retained 72 and 40% of its activity after 6 h at pH 9 and pH 10, respectively. After separation by isoelectric focusing electrophoresis, a zymogram indicated one major FAE activity exhibiting pI value of 10.5. This revised version was published online in August 2006 with corrections to the Cover Date.     

A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene

An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity. Keywords: Bacter     

High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

Summary Tissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.     

Organic solvent tolerance of an alkaline protease from salt-tolerant alkaliphilic <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> strain Mit-1

A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60–80°C and displayed optimum activity at 70°C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.     

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.     

High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization

The extracellular lipase gene from Yarrowia lipolytica (YlLip2) was cloned into the pPICZalphaA and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The lipase was successfully expressed and secreted with an apparent molecular weight of 39kDa using Saccharomyces cerevisiae secretion signal peptide (alpha-factor) under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). The lipase activity of 12,500,000U/l (2.10g total protein and 0.63g lipase per liter) was obtained in a fed-batch cultivation, where methanol feeding was linked to the dissolved oxygen content after initial glycerol culture. After fermentation, the supernatant was concentrated by ultrafiltration with a 10kDa cut off membrane and purified with ion exchange chromatography using Q Sepharose FF. Deglycosylation showed that the recombinant lipase is a glycoprotein which contains the same content of sugar (about 12%) as the native lipase from Y. lipolytica. The optimum temperature and pH of the recombinant lipase was 40 degrees C and 8.0, respectively. The lipase showed high activity toward long-chain fatty acid methyl esters (C12-C16).     

Purification and partial characterization of β-1,3-glucanase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>

A thermostable extracellular β-1,3-glucanase from Chaetomium thermophilum was purified to homogeneity by fractional ammonium sulfate precipitation, Pheny1-Sepharose hydrophobic interaction chromatography, ion exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-100. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 76.3 kDa. The enzyme exhibited optimum catalytic activity at pH 6.0 and 60 °C. It was thermostable at 50 °C, and retained 90% activity after 60 min at 60 °C. The half-life at 65 °C, 70 °C and 80 °C was 55 min, 21.5 min, and 5 min, respectively. The N-terminal amino acid sequence (8 residues) of the enzyme was HWLGDIPH. The HPLC analysis showed that the only enzymatic product formed from laminarin by the purified β-1,3-glucanase was glucose, indicating that the enzyme is an exo-β-1,3-glucanase (EC 3.2.1.58).