Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Purification and characterization of a cold active alkaline protease from <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp., isolated from Kashmir,India

A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature range of 4–37°C however, showed optimum growth at 15°C. The strain was found to over-produce proteases when it was grown in media containing lactose as carbon source (157.50 U mg⁻¹). The maximum specific enzyme activity (398 U mg⁻¹) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea, ammonium chloride and ammonium sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg⁻¹. The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be ~55 kDa by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5 to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15°C. Proteolytic activity reduced gradually with higher temperatures with a decrease of 56% at 40°C. The purified enzyme was checked for the removal of protein containing tea stains using a silk cloth within a temperature range of 10–60°C. The best washing efficiency results obtained at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are vulnerable to high temperatures.     

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

Use of response surface methodology to optimize protease synthesis by a novel strain of Bacillus sp. isolated from Portuguese sheep wool

Aims: To investigate the influence of yeast extract, peptone, temperature and pH upon protease productivity by Bacillus sp. HTS102 – a novel wild strain isolated from wool of a Portuguese sheep breed (Merino). Methods and Results: A 2⁴ full factorial, central composite design together with response surface methodology was used to carry out the experiments and analyse the results, respectively. Among the individual parameters tested, temperature and peptone concentration produced significant effects upon protease productivity. A high correlation coefficient (R² = 0·994, P < 0·01) indicated that the empiric second‐order polynomial model postulated was adequate to predict said productivity, with the optimum loci characterized by: temperature of 43°C, peptone content of 1·4 g l^?1, pH of 5·1 and yeast extract concentration of 10·0 g l^?1. Conclusions: Protease synthesis depends chiefly on temperature and peptone level. The maximum protease activity was more than twice that obtained with the basal medium, so the experimental design and analysis undertaken were effective towards process optimization. Significance and Impact of the Study: Rational choice of processing conditions for maximum protease productivity will be relevant if an economically feasible fermentation process based on Bacillus sp. HTS102 is intended.     

A statistical approach for the enhanced production of thermostable alkaline protease showing detergent compatibility activity from Bacillus circulans

Abstract

Response surface methodology (RSM) was employed to enhance the production of a thermostable alkaline protease from Bacillus circulans. Significant influences of peptone, yeast extract, and glucose on protease production were noted with a one-variable-at-a-time optimization strategy. Then, a full factorial central composite design (CCD) was applied to study the effects of glucose, peptone, and yeast extract to determine the optimal concentrations of these compounds for protease production by B. circulans under shake flask fermentation conditions. The statistical reliability and significance of the model was validated by an F-test for analysis of variance (ANOVA); enzyme production was improved significantly under optimized conditions. The enzyme was purified by ammonium sulphate fractionation, and gel filtration chromatography. Maximum enzyme activity was observed at 60°C temperature, and at pH 10. Alkaline protease from B. circulans showed excellent compatibility and stability in the presence of commercial detergents like Ariel, Surf Excel, Tide, Rin, Nirma, Wheel, and Doctor and showed excellent blood destaining effectiveness with commercial detergents.     

Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation

This paper reports the production of a cellulase-free and alkali-stable xylanase in high titre from a newly isolated Bacillus pumilus SV-85S using cheap and easily available agro-residue wheat bran. Optimization of fermentation conditions enhanced the enzyme production to 2995.20 ± 200.00 IU/ml, which was 9.91-fold higher than the activity under unoptimized basal medium (302.2 IU/ml). Statistical optimization using response-surface methodology was employed to obtain a cumulative effect of peptone, yeast extract, and potassium nitrate (KNO₃) on enzyme production. A 2³ central composite design best optimized the nitrogen source at the 0 level for peptone and yeast extract and at the −α level for KNO₃, along with 5.38-fold increase in xylanase activity. Addition of 0.1% tween 80 to the medium increased production by 1.5-fold. Optimum pH for xylanase was 6.0. The enzyme was 100% stable over the pH range from 5 to 11 for 1 h at 37°C and it lost no activity, even after 3 h of incubation at pH 7, 8, and 9. Optimum temperature for the enzyme was 50°C, but the enzyme displayed 78% residual activity even at 65°C. The enzyme retained 50% activity after an incubation of 1 h at 60°C. Characteristics of B. pumilus SV-85S xylanase, including its cellulase-free nature, stability in alkali over a long duration, along with high-level production, are particularly suited to the paper and pulp industry.     

Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum)

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20?mM tris-CaCl₂ buffer (pH 8.2) with a flow rate of 0.5?mL min^?1. The molecular weight and purity of ～23?kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697?U?mg^?1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

Production of laccase byCurvularia sp.

ACurvularia sp. isolated from soil was found to contain laccase activity toward guaiacol as substrate. The organism produced an extracellular laccase in a medium containing yeast extract, peptone and dextrose. Initial medium pH 4.0 and cultivation temperature 30°C were found to be most suitable for maximum enzyme production. The optimum pH and temperature for laccase activity were found to be 5.2 and 50°C, respectively. Under optimum conditions, the enzyme had aK _m (guaiacol) of 0.75 mmol/L and aV of 1.50 CU min⁻¹ ml⁻¹. Some divalent metal ions inhibited laccase activity at very low concentrations.     

The biotechnological potential of subtilisin‐like fibrinolytic enzyme from a newly isolated Lactobacillus plantarum KSK‐II in blood destaining and antimicrobials

An antimicrobial oxidative‐ and SDS‐stable fibrinolytic alkaline protease designated as KSK‐II was produced by Lactobacillus plantarum KSK‐II isolated from kishk, a traditional Egyptian food. Maximum enzyme productivity was obtained in medium containing 1% lactose and 0.5% soybean flour as carbon and nitrogen sources, respectively. Purification of enzyme increased its specific activity to 1,140‐fold with a recovery of 33% and molecular weight of 43.6 kDa. Enzyme activity was totally lost in the presence of ethylenediaminetetraacetic acid and was restored after addition of Fe²⁺ suggesting that KSK‐II is a metalloprotease and Fe²⁺ acts as cofactor. Enzyme hydrolyzed not only the natural proteins but also synthetic substrates, particularly Suc‐Ala‐Ala‐Pro‐Phe‐pNA. KSK‐II can hydrolyze the Lys‐X easier than Arg‐X; thus, it was considered as a subtilisin‐family protease. Its apparent K_m, V_max, and K_cat were 0.41 mM, 6.4 µmol mg^?1 min^?1, and 28.0 s^?1, respectively. KSK‐II is industrially important from the perspectives of its maximal activity at 50°C (stable up to 70°C), ability to function at alkaline pH (10.0), stability at broad pH ranges (7.5–12.0) in addition to its stability toward SDS, H₂O₂, organic solvents, and detergents. We emphasize for the first time the potential of fibrinolytic activity for alkaline proteases used in detergents especially in blood destaining. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:316–324, 2015     

Chitinolytic enzymes from the newly isolated <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> SBK1: study of the mosquitocidal activity

Aeromonas hydrophila SBK1 (GenBank accession no. HM802878.1), a potent chitinolytic bacterium, was isolated from a pool of 30 chitinolytic isolates. The isolate showed higher chitinolytic activity in respect to clear zone to colony size ratio of 2.15. Maximum production of chitinolytic enzymes, viz., β-N-acetyl-glucosaminidase and chitinase (specific activity 655.3 and 71.6 U mg⁻¹, respectively) by A. hydrophila SBK1 was observed in the synthetic media, containing (w/v)-colloidal chitin, 4.0%; peptone, 0.3%; phosphate, 0.3% (0.15% of each KH₂PO₄ and K₂HPO₄); NaCl, 0.25%; MgSO₄, 0.05%; KCl, 0.05%; pH 7.0 and at 35°C after 72 h of incubation. Both carbon-to-nitrogen (C/N) and carbon-to-phosphate (C/P) ratio of 13.33 were found optimum for chitinase production. Enzyme productivity increased about twofold in optimized culture condition in respect to its un-optimized state. The crude enzyme showed optimum activity against Culex quinquefasciatus larvae in native water at pH 7.0 and 35°C (LD₅₀ 0.60 U ml⁻¹ at 48 h). Therefore, the studied chitinases can be used as an effective mosquitocidal agent.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

Production,characterization, and immobilization of partially purified surfactant–detergent and alkali-thermostable protease from newly isolated Aeromonas caviae

Proteolytic Aeromonas caviae P-1-1 growing at wide-ranging pH (7.0–11.0) and moderate salinity (0–5% NaCl) was isolated from cattle shed of Thanjavur, India. It produced lipase, gelatinase, and polyhydroxybutyrate. Different culture conditions, incubation time, carbon and nitrogen sources, vitamins, amino acids, surfactants, and metal ions for optimal growth and protease production of P-1-1 were examined. Maximum protease (0.128?U/mL) production was achieved with 1% fructose, 1% yeast extract, 0.1% ammonium sulfate, 3% NaCl, 0.1% CaCl₂?·?2H₂O, 1% glycine, 0.1% vitamin E, and 0.1% Tween-40 at pH 8.0 after 42?hr of incubation at 37°C. It was active over broad range of pH (7.0–12.0), temperature (15–100°C), and salinity (0–9% NaCl) with optima at pH 10.0, 55°C, and 3% NaCl. It retained 65 and 48% activities at pH 12.0 and 100°C, respectively. Partially purified protease was highly stable (100%) within pH range 7.0–12.0 and salinities of 0–5% NaCl for 48?hr. Cu²⁺, Mn²⁺, Co²⁺, and Ca²⁺ did not inhibit its activity. Its stability at extreme pHs, temperatures, and in the presence of surfactants and commercial detergents suggests its possible application in laundry detergents. Partially purified protease was immobilized and reused. This is the first report of alkali-thermotolerant, surfactant–detergent-stable partially purified extracellular protease from A. caviae.     

Enhanced production of an alkaline pectinase from Streptomyces sp. RCK-SC by whole-cell immobilization and solid-state cultivation

An alkalophilic Streptomyces sp. RCK-SC, which produced a thermostable alkaline pectinase, was isolated from soil samples. Pectinase production at 45 °C in shaking conditions (200 rev min⁻¹) was optimal (76,000 IU l⁻¹) when a combination of glucose (0.25% w/v) and citrus pectin (0.25% w/v) was added along with urea (0.25% w/v) in the basal medium devoid of yeast extract and peptone. All the tested amino acids and vitamins greatly induced pectinase production and increased the specific productivity of pectinase up to 550%. In an immobilized cell system containing polyurethane foam (PUF), the pectinase production was enhanced by 32% (101,000 IU l⁻¹) compared to shake flask cultures. In solid-state cultivation (SSC) conditions, using wheat bran as solid substrate, pectinase yield of 4857 IU g⁻¹ dry substrate was obtained at substrate-to-moisture ratio of 1:5 after 72 h of incubation. The partially purified pectinase was optimally active at 60 °C and retained 80% of its activity at 50 °C after 2 h of incubation. The half life of pectinase was 3 h at 70 °C. Pectinase was stable at alkaline pH ranging from 6.0 to 9.0 for more than 8 h at room temperature retaining more than 50% of its activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Production of different proteases from fish gut microflora utilizing tannery fleshing

Three alkaline protease‐producing strains designated as ANFLR1, NPLR1, and PROLR15 were isolated from Labeo rohita fish gut. These strains are able to produce alkaline protease using tannery fleshing (TF) as the sole carbon and nitrogen source and were identified as Bacillus megaterium, Serratia marcescens, and novel Pontibacter sps. Proteases from these organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, and column chromatography. SDS‐PAGE revealed molecular weights of the proteases to be 46 kDa (ANFLR1), 52 kDa (NPLR1), and 58 kDa (PROLR15). The optimum pH and temperature for the protease activity of ANFLR1, NPLR1, and PROLR15 were found to be 10.5, 11.5, 9, and 70^°C, 60^°C, and 50^°C, respectively. The maximum protease activities at the optimum conditions were 420 U/mL (ANFLR1), 550 U/mL (NPLR1), and 530 U/mL (PROLR15). Inhibition of the NPLR1 protease by pepstatin confirmed aspartate‐type enzymatic activity. Fe³⁺ enhanced the activity of PROLR15 protease. Unlike all other microbial proteases known so far, the PROLR15 enzyme did not require Ca²⁺ for activity and thermal stability. SDS‐PAGE and scanning electron microscopy analyses confirmed the conversion of high molecular weight substrate (TF) to low molecular weight peptides by these proteases. The alkaline metalloprotease production by novel Pontibacter sps. and aspartate protease production by S. marcescens remain unexplored. Hence, TF with its relatively abundant availability can be beneficially utilized for alkaline protease production through the fish gut microbial fermentation processes.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.