Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

A novel nonionic surfactant- and solvent-stable alkaline serine protease from <Emphasis Type="Italic">Serratia</Emphasis> sp. SYBC H with duckweed as nitrogen source: production,purification, characteristics and application

A novel nonionic surfactant- and hydrophilic solvent-stable alkaline serine protease was purified from the culture supernatant of Serratia sp. SYBC H with duckweed as nitrogen source. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH range between 5.0 and 11.0, with the maximum activity at pH 8.0. It is also fairly active over the temperature range between 30 and 80°C, with the maximum activity at 40°C. The protease activity was substantially stimulated by Mn²⁺ and Na⁺ (5 mM), up to 837.9 and 134.5% at 40°C, respectively. In addition, Mn²⁺ enhanced the thermostability of the protease significantly at 60°C. Over 90% of its initial activity remained even after incubating for 60 min at 40°C in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH. The protease retained 81.7, 83.6 and 76.2% of its initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease. Washing experiments revealed that the protease has an excellent ability to remove blood stains.     

Purification and Characterization of an Extracellular Cold-Active Serine Protease from the Psychrophilic Bacterium <Emphasis Type="Italic">Colwellia</Emphasis> sp. NJ341

Colwellia sp. NJ341, isolated from Antarctic sea ice, secreted a cold-active serine protease. The purified protease had an apparent Mr of 60 kDa by SDS-PAGE and MALDI-TOF MS. It was active from pH 5–12 with maximum activity at 35 °C (assayed over 10 min). Activity at 0 °C was nearly 30% of the maximum activity. It was completely inhibited by phenylmethylsulfonyl fluoride.     

Xylanase production under solid-state fermentation and its characterization by an isolated strain of <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> in India

Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like K_m and V_max were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl₂, xylose and Tween 80, while significantly inhibited by Hg⁺⁺, Cu⁺⁺ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca⁺⁺. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Isolation and screening of Streptomyces sp. Al-Dhabi-49 from the environment of Saudi Arabia with concomitant production of lipase and protease in submerged fermentation

In this study, Streptomyces sp. Al-Dhabi-49 was isolated from the soil sample of Saudi Arabian environment for the simultaneous production of lipase and protease in submerged fermentation. The process parameters were optimized to enhance enzymes production. The production of protease and lipase was found to be maximum after 5 days of incubation (139.2 ± 2.1 U/ml, 253 ± 4.4 U/ml). Proteolytic enzyme increases with the increase in pH up to 9.0 (147.2 ± 3.6 U/ml) and enzyme production depleted significantly at higher pH values. In the case of lipase, production was maximum in the culture medium containing pH 8.0 (166 ± 1.3 U/ml). The maximum production of protease was observed at 40 °C (174 ± 12.1 U/ml) by Streptomyces sp. Lipase activity was found to be optimum at the range of temperatures (30–50 °C) and maximum production was achieved at 35 °C (168 ± 7.8 U/ml). Among the evaluated carbon sources, maltose significantly influenced on protease production (218 ± 12.8 U/ml). Lipase production was maximum when Streptomyces sp. was cultured in the presence of glucose (162 ± 10.8U/ml). Among various concentrations of peptone, 1.0% (w/v) significantly enhanced protease production. The lipase production was very high in the culture medium containing malt extract as nitrogen source (86 ± 10.2 U/ml). Protease production was maximum in the presence of Ca²⁺ as ionic source (212 ± 3.8 U/ml) and lipase production was enhanced by the addition of Mg²⁺ with the fermentation medium (163.7 ± 6.2 U/ml).     

Purification and characterization of a Bacillus sp. SAM1606 thermostable α-glucosidase with transglucosylation activity

We purified a novel -glucosidase to homogeneity from an Escherichia coli recombinant transformed with the -glucosidase gene from thermophilic Bacillus sp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known -glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety of O--d-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (k _O) and the Michaelis constant (K _m) values at 55°C and pH 6.0 for sucrose were 54.6 s^–1 and 5.3 mm, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75°C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8 m sucrose at 60°C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65°C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

Characterization of an extracellular glycoprotein inhibitor of trypsin (TI-23) produced by Streptomyces sp. 23

Summary The production of an extracellular trypsin inhibitor, TI-23, was found to parallel the growth of Streptomyces sp. 23 at different cultivation temperatures, reaching a maximum level at late exponential phase. Although the different temperatures (18°, 28° and 37°C) did not greatly affect the growth of the microorganism, they proved to be an important factor for extracellular inhibitory activity. Maximum specific rates of both cell growth and production of the inhibitor were recorded during the cultivation of Streptomyces sp. 23 at 37°C. TI-23 proved to be a monomeric glycoprotein containing 17% carbohydrate and differing in amino acid composition from the known extracellular proteinase inhibitors of streptomycetes. The molecular mass of the inhibitor was estimated to be about 13 kDa and the isoelectric point 4.3. The inhibition spectrum of TI-23 included trypsin as well as some microbial alkaline proteinases.     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

A new thermolabile alkaline phospholipase D from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS628

A phospholipase D (PLD₆₂₈), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7 ∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD₆₂₈ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD₆₂₈ lacks the transphosphatidylation activity. PLD₆₂₈ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.     

Production and characterization of a thermostable protease produced by an asporogenous mutant ofBacillus stearothermophilus

Summary An asporogenous mutant ofBacillus stearothermophilus (TPM-8) which produces 4-fold higher levels of a thermostable neutral protease than does wild-type strain 308-1 was obtained by mutagenesis with ethyl methanesulfonate. The protease produced by both the mutant and wild-type strain is a metalloprotease requiring Zn²⁺ and Ca²⁺ for activity and thermostability, respectively. It has a temperature optimum of 80°C at pH 7.0 and is highly thermostable, retaining 60% of its activity after 60 min at 85°C. The properties of the enzyme are similar to those of thermolysin.     

Preparation of vinyl thymidine ester catalyzed by protease and its chemical polymerization

Transesterification reaction of 0.25 M thymidine with 1 M divinyladipate in dimethylformamide (DMF) was catalyzed by an alkaline protease (5 mg ml^–1) from Streptomyces sp. (20 units mg^–1 min) at 30 °C for 7 days to give 5-O-vinyladipoyl thymidine (yield 77%) without formation of any by-products. Poly(vinyl alcohol) containing thymidine branches could be obtained by its free-radical polymerization.     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.     

Purification and Characterization of a Thermo- and Organic Solvent-Tolerant Alkaline Protease from Bacillus sp. JER02

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. K _m and V_max of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H₂O₂ (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.     

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH₄)₂SO₄ and soybean flour as nitrogen sources and KH₂PO₄ at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

Isolation of thermostable D-hydantoinase-producing thermophilicBacillus sp SD-1

Summary A thermophilic bacterium which showed highest thermostability and activity of the hydantoinase was isolated from 1m000 thermophiles and identified to beBacillus sp. SD-1 according to morphological and physiological characteristics. The optimal growth temperature of the bacterium was about 60°C. The hydantoinase ofBacillus sp. SD-1 was strictly D-specific, and optimal pH and temperature were determined to be about 8.0 and 70°C, respectively. The D-hydantoinase was stable upto 70°C, and half-life of the enzyme was about 20 min at 80°C.     

Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast

A newly isolated halotolerant Bacillus sp. VITP4 was investigated for the production of extracellular protease. 16S rRNA gene analysis identified it as Bacillus aquimaris. Enzyme secretion corresponded with growth (G_t, 38 min) in the basal Zobell medium, reaching a maximum during stationary phase (630 U/ml, 48 h). Protease production was investigated in different salt concentrations (0–4 M). While growth was optimum in the basal medium, higher levels of protease activity were observed in 0.5 M salt medium (728 U/ml, 48 h) and 1 M salt medium (796 U/ml, 78 h) with 21% and 32% increase in production, respectively. Salt concentrations above 2.5 M did not support bacterial growth. The optimum pH and temperature for production were pH 7.5 and 37 °C, respectively. A combination of peptone and yeast extract yielded optimum protease secretion. Inorganic nitrogen sources proved to be less favourable. Production was reduced in the presence of readily available carbon sources owing to catabolic repression. Effect of various salts (1–6%) indicated favourable bacterial growth in these conditions for producing proteolytic molecules with increased activity. The study assumes significance in the ability of the halotolerant bacterium to survive in a wide range of salinity and yield optimum levels of extracellular protease.