A thermostable,alkaline-active,keratinolytic proteinase from chrysosporium keratinophilum

Thermostable alkaline proteinase was produced by a strain of Chrysosporium keratinophilum when cultured in lactose/mineral salt medium incorporating keratin solubilized with DMSO. The proteinase, partially purified by cold-acetone precipitation followed by gel-filtration on Sephadex G-75, was optimally active at pH 9 and stable from pH 7 to 10 with over 90% relative residual activity after incubation at 25°C for 24 h. The optimum temperature for enzyme activity was 90°C at which the activity half-life was 30 min. Enzyme activity was stimulated by Fe²⁺ and inhibited by 1,10 o-phenanthroline. Gel-filtration indicated an M _r of 69 kDa.The authors are with the Department of Microbiology, Faculty of Biological Sciences, P.M. B.006, University of Nigeria, Nsukka, Enugu State, Nigeria     

Partial purification and some properties of an alkaline cellulase from an alkalophilic Bacillus sp.

A newly isolated Bacillus species, which grew optimally at 30°C and pH 10, produced a carboxymethylcellulase in a medium containing 10 g CM-cellulose/l. The enzyme, when partially purified by gel filtration, had a mass of about 29 kDa as determined by both SDS-PAGE and gel filtration chromatography. It was optimally active at pH 9.5 and 40°C, and was stable from pH 7 to 11 at 4°C for 24 h. The enzyme was stimulated by Ca²⁺ (1mm) but was completely inhibited by Hg²⁺ (1mm). Neither EDTA nor EGTA (10mm) affected the activity.The author is with the Department of Biological Sciences, University of Jordan. PO Box 2686, Amman 11181, Jordan     

Protein enrichment of lignocellulose resulting from the growth of two Streptomyces strains

Two Streptomyces strains were grown on sugarcane bagasse and groundnut hulls lignocelluloses in semi-solid state culture at 37°C for 12 weeks. Best results gave a 45% depletion of sugarcane bagasse lignocellulose with a 21% crude protein content of final material. The possibility of using S. viridosporus to improve the protein content of both lignocelluloses for use as an animal feedstock supplement is discussed.At the time of this research the authors were with the Department of Biological Sciences, Rivers State University of Science & Technology, PMB 5080, Port Harcourt, Nigeria and the Department of Biological Sciences, University of Calabar, PMB 1115, Calabar, Nigeria. Dr lyo is now with the Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.     

Thermotolerant strain of Bacillus licheniformis producing lipase

A thermotolerant variant of Bacillus licheniformis strain H1 (isolated from Jordan valley soil), was highly active in degrading macromolecules, and possessed a lipase activity with a half life of 30 min at 70°C. This activity was produced during exponential growth. The extracellular crude lipase showed maximal activity at pH 10, and retained 65% of its stability at pH 12.The author is with the Department of Biological Sciences, University of Jordan, P.O. Box 2686, Amman 11181, Jordan     

Production of a thermophilic,extracellular alkaline protease by Bacillus stearothermophilus AP-4

A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mm CaCl₂. The enzyme was completely inactivated by PMSF, EDTA and -mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India     

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.     

Purification and characterization of a thermodynamic stable serine protease from Aspergillus fumigatus

A thermostable extracellular serine protease from Aspergillus fumigatus was purified 8.8-fold using a 4-step protocol. The enzyme was produced using a 36 h solid-state culture, had a molecular weight of 88 kDa and exhibited maximal enzyme activity at pH 7 and 60 °C. Structural analysis revealed that the protease is monomeric and non-glycosylated. Thermal inactivation of the pure enzyme followed first-order kinetics. The half-life (t_1/2) of the pure enzyme at 50, 60 and 70 °C was 65, 34 and 14 min, respectively. The denaturation and activation energies were 69 and 62 kJ mol⁻¹, respectively. Thermodynamic parameters (entropy and enthalpy) suggested that the protease was highly thermostable. This is the first report on the thermodynamic parameters of proteases produced by A. fumigatus.     

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.     

Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co²⁺ and Hg²⁺, whereas Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺ and Sr²⁺ had little or no inhibitory effect. However, Mn²⁺ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t _1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca²⁺. Correspondence to: A. B. Salleh     

Chilling, oxidative stress and antioxidant responses in shoot cultures of rice

Chilling of shoot cultures from Oryza sativa L. cv. Taipei 309, to 4 °C leads to conditions of oxidative stress. Tissue H₂O₂ was observed to increase more than fourfold by 8 d of chilling, and levels of reduced glutathione, which normally rise in growing shoot cultures at 25 °C, were considerably repressed in chilled cultures. Whilst the activity of ascorbate peroxidase in chilled shoots remained similar to the activities in control cultures at 25 °C, the most notable effects of chilling to 4 °C were the very significant loss of catalase and glutathione reductase activity. Although prior exposure of shoot cultures to abscisic acid (ABA) at 25 °C increased levels of catalase activity, such increased levels were not sustained when the pre-treated cultures were placed at 4 °C. Moreover such pre-treatment with ABA did not increase the subsequent ability of shoot cultures to grow at 4 °C.Abbreviations GSH reduced glutathione - GSSG oxidised glutathione - ABA cis-abscisic acid This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu)

Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.     

Comparative studies of a new microbial bate and the commercial bate ‘Oropon’ in leather treatment

An extracellular protease ofBacillus subtilis K2, when compared with the pancreatic bate Oropon used at comparable protease activity, produced a finer grain and greater elasticity of goat pelt. Quality of the processed, finished leather after microbial bating showed <41% and <14% increases in tensile and tear strengths, respectively, compared with Oropon-bated leather.     

Production and characterization of a xylanase from Cyathus stercoreus

Cyathus stercoreus grown on wheat straw had a higher xylanase activity than when it was grown on rice husk or extracted hemicellulose. Inclusion of casein hydrolysate, Tween 80 and Mn²⁺ (at 0.02%, 0.2% and 0.075%, respectively) increased the production of extracellular xylanase. Optimal yield of xylanase (0.73 U/ml) was at pH 5.6 after 9 to 12 days at 30°C. The xylanase was stable at pH 4.5 to 7.5 for 2h but above 50°C its stability fell sharply.The authors are with the Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India;     

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.     

Seasonal variation in the physical properties of agar and biomass of Gracilaria sp. (chorda type) from Tosa Bay,southern Japan

Plants of Gracilaria sp.(chorda type), which grow along the coast of Uranouchi Inlet in Tosa Bay, southern Japan, showed the highest biomass in the summer (26 °C to 31 °C) and spring season (15.1 °C to 24.9 °C). Maximum biomass was 6952 g m^–2 in July, but gradually decreased in the autumn (30.5 °C in September to 20 °C in November) and winter (19.5 °C in December to 14.9 °C in February). Variation in yields and gel strength of the agars, were shown to depend on the time in the season. After alkali treatment (5% NaOH, 2 h) at three different temperatures (70, 80, and 90 °C), the agars showed gel strengths essentially that of commercial grade agars, with the best gel obtained at 80 °C. Maximum gel strength (1455 g cm^–2 of 1.5% agar gel) occurred in winter when the biomass and agar yield were low. Minimum gel strength was in spring. Gel strength was inversely correlated with agar yield, but was positively correlated with apparent viscosity. Maximum viscosity was 40 cP. in December. Gelling temperatures, pH of 1.5% agar gel, and moisture content in agars showed little variation.     

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.     

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.     

Medium optimization for the production of glucose isomerase from thermophilic Streptomyces thermonitrificans

A thermophilic strain of Streptomyces thermonitrificans produced a high activity of intracellular glucose isomerase (12 U/ml) when grown in a medium containing 1% (w/v) xylose, supplemented with 2% (w/v) sorbitol as the second carbon source, at 50°C for 16 h. Addition of Mg²⁺ enhanced enzyme production but the organism could grow and produce the enzyme in the absence of Co²⁺.The authors are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, IndiaNCL Communication No. 5813     

Production of neutral and alkaline extracellular proteases by the thermophilic fungus, Scytalidium thermophilum, grown on microcrystalline cellulose

Extracellular proteases produced by Scytalidium thermophilum, grown on microcrystalline cellulose, were most active at pH 6.5–8 and 37–45 °C when incubated for 60 min. Highest protease activity was at day 3 where endoglucanase activity was low. Protease activity measurements with and without the protease inhibitors, p-chloromercuribenzoate, PMSF, antipain, E-64, EDTA and pepstatin A, suggest production of thiol-containing serine protease and serine proteases. Endoglucanase and Avicel-adsorbable endoglucanase activity in culture medium was not significantly affected by protease inhibitors.