Thermostable alkaline lipase from a newly isolated thermophilic Bacillus,strain A30-1 (ATCC 53841)

A thermophilic bacterium was isolated from a hot spring area of Yellowstone National Park. The organism grew optimally at 60–65°C and in the pH range of 6–9. It was characterized as Bacillus sp. In the presence of corn or olive oil (1.0%) as the growth substrate, this Bacillus produced an extracellular lipolytic activity (EC 3.1.1.3). The enzyme activity could be efficiently recovered by ultrafiltration of cell-free culture supernatant. The partially purified lipase preparation had an optimum temperature of 60°C, at an optimum pH of 9.5. It retained 100% of the original activity after being heated at 75°C for half an hour. The half life of the enzyme was 8 h at 75°C. The enzyme retained at least 90% of the original activity after it was incubated at 60°C for 15 h at pH's in the range of 5 to 10.5. The enzyme was active on triglycerides containing fatty acids having a carbon chain length of C16 : 0 to C22 : 0 as well as on natural fats and oils. The enzyme activity was stable to both hydrogen peroxide and alkaline protease which are detergent ingredients. The purified enzyme had an isoelectric point of 5.15 and an approximate molecular weight of 65,000.     

Production of alkaline protease in a low-cost medium by alkalophilic Bacillus sp. and properties of the enzyme

The main object of this research was to obtain a large amount of alkaline protease in a low-cost medium, for which purpose Bacillus sp. B21-2 was isolated from soil. The enzyme production by this strain reached the maximum level of 15,000 u/ml in a short cultivation time of 24–28 h in alkaline medium consisting of cheap materials such as soybean meal and bonito extract. The optimum pH and temperature for activity of the purified enzyme were 11.5 and 60°C respectively. The enzyme was stable up to 50°C but inactivated after 10 min at 60°C. The enzyme activity was completely inhibited by diisopropylfluorophosphate.     

Purification and Characterization of an Extracellular Low Temperature-Active and Alkaline Stable Peptidase from Psychrotrophic Acinetobacter sp. MN 12 MTCC (10786)

An extracellular low temperature-active alkaline stable peptidase from Acinetobacter sp. MN 12 was purified to homogeneity with a purification fold of 9.8. The enzyme exhibited specific activity of 6,540 U/mg protein, with an apparent molecular weight of 35 kDa. The purified enzyme was active over broad range of temperature from 4 to 60 °C with optimum activity at 40 °C. The enzyme retained more than 75 % of activity over a broad range of pH (7.0–11.0) with optimum activity at pH 9.0. The purified peptidase was strongly inhibited by phenylmethylsulfonyl fluoride, giving an indication of serine type. The K _m and V _max for casein and gelatin were 0.3529, 2.03 mg/ml and 294.11, 384.61 μg/ml/min respectively. The peptidase was compatible with surfactants, oxidizing agents and commercial detergents, and effectively removed dried blood stains on cotton fabrics at low temperature ranging from 15 to 35 °C.     

An organic solvent-, detergent-, and thermostable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45°C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over a broad range of temperatures (45–70°C) and pH (8–10) range with an optimum activity at pH 10 and 65°C. It was comparatively stable in the presence of a relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45°C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.     

Characterization of an alkaline elastase from alkalophilic Bacillus Ya-B

The alkaline elastase produced by alkalophilic Bacillus Ya-B was a new type of proteinase which had a very high optimum pH and high elastolytic activity. It also had a high hydrolyzing activity against keratin and collagen. The molecular weight was determined to be 23 700 and 25 000 by ultracentrifugation analysis and SDS-polycrylamide gel electrophoresis, respectively. The isoelectric point was 10.6. The optimum reaction temperature was 60°C. Like many alkaline proteinases, this enzyme required Ca²⁺ for stability. The optimum reaction pH was 11.75 toward casein and elastin-orcein. The K_cat/K_m values of the enzyme to synthetic substrates were constant from pH 8.5 up to 12.75. The enzyme was stable in the pH range 5.0–10.0. The enzyme was inhibited by alkaline proteinase inhibitors Streptomyces subtilisin inhibitor and microbial alkaline proteinase inhibitor, but not by elastatinal or the metalloproteinase inhibitor metalloproteinase inhibitor. Sodium chloride inhibited the elastolytic activity but not the caseinolytic activity at a concentration below 0.2 M. The inhibitory effect of sodium chloride to elastolytic activity was much more prominent at pH 9.0 than at pH 11.5. More than 50% of the enzyme bound onto elastin in the pH range below the isoelectric point of this enzyme. The amino-terminal sequence of the enzyme was determined, and compared with those of subtilisin BPN′ and subtilisin Carlsberg. Extensive sequence homology was noted among these three enzymes.     

Production and Properties of Alkaline Dextranase from a Newly Isolated Brevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.

Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.     

Immobilization of β-amylase on polystyrene cation exchange resin equilibrated with Al3+ions (IR-120 Al3+)

A simple procedure for the immobilization of β-amylase (EC3.2.1.2) on IR-120 Al³⁺ is described. Immobilization brought about a slight shift in the optimum pH towards the alkaline side relative to that of the soluble enzyme. Immobilized β-amylase showed a broader temperature optima (45–55°C) compared with the soluble enzyme (45°C). A six-fold increase in the K_m was also noticed. On storage and repeated use the immobilized enzyme retained approximately 60% of its initial activity up to 120 days at room temperature.     

Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil

A new CGTase was obtained from Bacillus firmus, strain 7B, isolated from oat soil culture, using a high alkaline pH medium containing 1% Na₂CO₃. The enzyme was characterized in soluble form, for pH 5–11, temperature from 30 to 85 °C, using a 1% maltodextrin substrate solution and appropriate buffers. It produced mainly β-CD and the cell-free supernatant had a precipitating activity measured by the trichloroethylene method that is a 100-fold greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. The molecular weight of the pure protein was measured as 56,230 Da with SDS-PAGE. The optimum temperature for the enzyme activity was 50 °C and it was most active at pH 6.0. Thermal deactivation was noticeable above 65 °C and the enzyme was highly stable below 60 °C. The influence of substrate or product concentration on the initial rate of CD production was studied and the kinetic parameters were determined. The enzyme showed cyclization activity on different raw and hydrolyzed starches and hydrolyzed cornstarch gave the highest activity.     

Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800

The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN₃ and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.     

A novel organic solvent-stable alkaline protease from organic solvent–tolerant Bacillus licheniformis YP1A

An organic solvent-stable alkaline protease producing bacterium was isolated from the crude oil contaminant soil and identified as Bacillus licheniformis. The enzyme retained more than 95% of its initial activity after pre-incubation at 40 °C for 1 h in the presence of 50% (v/v) organic solvents such as DMSO, DMF, and cyclohexane. The protease was active in a broad range of pH from 8.0 to 12.0 with the optimum pH 9.5. The optimum temperature for this protease activity was 60 °C, and the enzyme remained active after incubation at 50–60 °C for 1 h. This organic solvent-stable protease could be used as a biocatalyst for organic solvent-based enzymatic synthesis.     

Production of alkaline protease by an alkaliphilic bacteria isolated from an alkaline soda lake

A new alkaliphilic strain of Microbacterium producing an alkaline protease was isolated from an alkaline soda lake in Ethiopia. High level of protease activity was produced in the presence of glucose and sucrose as carbon sources. The optimum temperature and pH for activity were 65°C and 9.5-11.5 respectively. Above 50°C, Ca²⁺ was required for enzyme activity and stability. At 55 and 60°C it retained 100 and 85% of its original activity respectively after 1 h incubation. The enzyme was stable over the pH range of 5-12. This revised version was published online in November 2006 with corrections to the Cover Date.     

Purification and properties of an acidic β-glucosidase from Acidobacterium capsulatum

Acidobacterium capsulatum, an acidophilic, mesophilic and chemoorganotrophic bacterium, produced an inducible, acidic β-glucosidase in the cellobiose medium. The enzyme was successively purified 109 times by CM-Sepharose, Sephacryl S-200 chromatography and preparative discontinuous polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the purified enzyme gave a single band at pH 4.3. The enzyme had an optimum pH of 3.0 and optimum reaction temperature of 55°C, being stable from pH 1.5 to 6.0 and at temperatures from 20 to 45°C. No activity was detected above pH 6.5 or above 65°C. The molecular weight of 90,000 was estimated by gel filtration and the enzyme had an isoelectric point of 7.0. The enzyme hydrolyzed aryl-β-glycosides and β-linked disaccharides.     

C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70 °C versus 60 °C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60–80 °C and 6.0–9.0 versus 40–60 °C and 5.0–8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 °C as compared to wild −type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.     

Screening Diverse Fungi for Laccases of Varying Properties

Qualitative screening of 295 fungi for laccases yielded 125 laccase positive ones, mostly basidiomycetes. Fifty of these were tested for laccase activity at pH 3.0, 4.5 and 6.0. Most showed maximum activity at pH 4.5, a few showed a broad activity range, two were optimal at pH 3.0 and only the mitosporic fungus Beltraniella sp. was best at pH 6. Most of the 25 fungi assayed at three different temperatures had an optimum at 45°C. The basidiomycete Auricularia sp. acted best at 30°C, while three others showed best activity at 60°C. This study shows the potential of screening diverse fungi for laccase with varying pH and temperature preferences for different applications.     

Biomass hydrolyzing enzymes from plant pathogen Xanthomonas axonopodis pv. punicae: optimizing production and characterization

Xanthomonas axonopodis pv. punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28 °C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55 °C while xylanase had highest activity at 45 °C. Heat treatment of enzyme extract at 75 °C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.     

Optimizing some factors affecting protease production under solid state fermentation

Production of extracellular alkaline protease by a locally isolated fungal species, Rhizopus oryzae, under solid state fermentation was optimized. The maximum enzyme activity under the optimum conditions of temperature (32?°C), relative humidity (90%–95%), spore count (～2?×?10⁵/g wheat bran), moisture content of solid substrate (140%) adjusted suitably with salt solution (M-9) of pH?5.5 was 341 unit/g wheat bran.     

Purification of keratinase from poultry farm isolate-Scopulariopsis brevicaulis and statistical optimization of enzyme activity

The fungus Scopulariopsis brevicaulis was isolated from poultry farm soil at Namakkal, India. The extracellular keratinase from this fungus was purified to homogeneity by ammonium sulphate precipitation and procedure involving DEAE-Cellulose and Sephadex G-100 chromatographic techniques. The purified enzyme was formed from a monomeric protein with molecular masses of 39 and 36 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 8.0 and the optimum temperature at pH 8.0 was 40 °C. The activity of purified keratinase with respect to pH, temperature and salt concentration was optimized by Box–Behnken design experiment. It was shown that a second-order polynominal regression model could properly interpret the experimental data with an R²-value of 0.9957 and an F-value of 178.32, based on the maximum enzyme activity examined. Calculated optimum conditions were predicted to confer a 100% yield of keratinase activity with 5 mM CaCl₂, pH 8.0 and at a temperature of 40 °C. The enzyme was strongly inhibited by PMSF, which suggests a serine residue at or near an active site. The purified keratinase was examined with its potential for dehairing the skin.     

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.     

Purification and characterization of a protease extracellularly produced by Monascus purpureus CCRC31499 in a shrimp and crab shell powder medium

Monascus purpureus CCRC31499 produced a protease when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine wastes. An extracellular protease was purified from the culture supernatant to homology. The protease had a molecular weight of 40,000 and a pI of 7.9. The optimal pH, optimum temperature, pH stability, and thermal stability of the protease were pH 7–9, 40 °C, pH 5–9, and 40 °C, respectively. In addition to protease activity, CCRC31499 also exhibited activity of enhancing vegetable growth in culture supernatant. This is also the first report of isolation of a protease from Monascus species.     

Enzymatic Properties of Newly Found Green Turtle Egg White Ribonuclease

Egg white ribonuclease was first found in green turtle eggs. The general properties were studied on substrate specificity, the optimum pH and temperature, and the effect of pH and temperature on the RNase activity. The enzyme studied was specific for poly (C) and degraded poly (U) at the lower rate and had the pH optimum at 7.0 and the optimum temperature at 40 °C. It was stable at alkaline range (pH 8.0–10.0) and up to 60 °C in pH 9.0 for 1 h, and unstable at acidic side for all temperatures. All of the properties studied showed similarity to RNase A. However, the optimum pH, broad range of optimum temperature and pH stability were different from RNase A. To evaluate the relationship of the structure and enzymatic properties, the 3D-structure of this enzyme was engineered by program MODELLER using two RNases (2BWL and 2BLZ) as starting models. The differences found in activity might be affected from the structure of micro environmental changing caused by amino acids deletion and substitution on the molecule.