Effects of deleting peptidoglycan hydrolase genes on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease / 生物工程学报

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangroves

Extracellular protease from culture supernatants of Streptomyces sp. LCJ12A was purified and characterized in thisstudy. The collected supernatant of Streptomyces sp. LCJ12A was purified by ammonium sulfate precipitation anddiethylaminoethyl cellulose chromatography. The molecular weight of the enzyme was 20.1 kDa. Sodium dodecylsulfate-polyacrylamide gel electrophoresis was used to determine the molecular weight of the enzyme. The culture filtrate,partially purified enzyme, and ammonium sulfate precipitate displayed catalysis and stability over pH 3–11.5 and showedoptimal activity at pH 10. The culture filtrate, ammonium sulfate precipitate, and partially purified enzyme were goodbetween 30 and 40°C and the optimum temperature was 35°C. The purified protease exhibited high catalytic activityand stability under different pH and temperature. The partially purified protease from Streptomyces sp. LCJ12A showeda substantial relative activity of 78% with bovine serum albumin (BSA), 43% with gelatin, and 96% relative activitywith casein. Lineweaver–Burk plot was used to calculate the Km and Vmax values for the partially purified protease.The Km values of Streptomyces sp. LCJ12A were 73.5 mM for casein, 44.54 mM for BSA, and 43.45 mM for gelatin.The Vmax values were 500 mM min−1 for casein, 303.03 mM min−1 for BSA, and 270.27 mM min−1 for gelatin fromStreptomyces sp. LCJ12A. The statistical analysis confirmed that compared to the other substrates, casein was significant.

Paenibacillus dendritiformis DDS2: A new member to alkaline-thermophilic protease producers

@#Aim: Most of the industrial processes require thermostable alkaline proteases. Thus, a search was initiated to isolate and characterize a bacterium which can produce thermostable alkaline protease. Methodology and results: Best higher titer thermostable alkaline protease producing wild type organism was screened from beef, dog and fish decaying soil samples. Among the 92 bacterial strains, three strains which produced alkaline proteases having activities at pH 10.5 and above 70 °C were selected. Among the three strains, the one from the dog decaying soil (Strain DDS2 ; DDS-dog decaying soil) which produced the protease showing highest activity at pH 10.5 and 73 °C and stability (half-life: 10.5 h) without additives was selected and identified. Based on the biochemical and morphological studies, strain DDS2 could be either Paenibacillus dendritiformis or P. thiaminolyticus. From 16S rDNA sequencing, the strain DDS2 was confirmed as P. dendritiformis. Conclusion, significance and impact: This is the first report published to show that P. dendritiformis is a protease producer and the organism was named as P. dendritiformis DDS2.

Characterization of a novel bidirectional promoter in Bacillus subtilis / 生物工程学报

Based on the transcriptome analysis data of a Bacillus licheniformis, a novel bidirectional promoter was identified from the strain and its transcriptional strength was analyzed. The expression level of a Bacillus clausii derived alkaline protease gene driven by the bidirectional promoter was studied by using the known strong constitutive promoter pShuttle-09 as a control. Three recombinant expression vectors and the corresponding recombinant bacteria were constructed. Under the control of the new promoter pLA and its reverse promoter pLB, the alkaline protease expression level respectively reached 164 U/mL and 111 U/mL. The results indicated that the transcription strength of pLA was significantly higher than that of pShuttle-09 and pLB, and both the pLA and pLB promoters could initiate the expression of the alkaline protease. Thus, it provides a new expression element for the heterogenous genes in Bacillus sp. and a new idea for the co-expression of two genes in one prokaryotic strain.

Levels of antioxidant enzymes and alkaline protease from pulp and peel of sunflower

Objective The activity of enzymes participating in the systems of antioxidant protection was assayed in the peel and pulp of sunflower. The essential roles of proteases in food stimulate research to find other sources of the enzyme especially from non-conventional sources. In the present work, we study several biochemical parameters in the pulp and peel of sunflower. Methods Pulp and peel of sunflower was extracted, antioxidant enzymes and non-enzymatic antioxidant were measured. Alkaline protease was measured and purified from pulp in sunflower. Results High carbohydrate concentration, beta-carotene, catalase and ascorbate peroxidase activities, free radical scavenging capacity and free flavonoid content were observed in the peel of sunflower. Whereas, MDA and ceruloplasmin activities were high in the pulp of sunflower. Conclusions The present study concluded that peel in sunflower are strong radical scavengers and can be considered as good sources of natural antioxidants for medicinal and commercial uses. Further analysis showed that protease activity was a significantly high in the pulp compared to the peel.

Levels of antioxidant enzymes and alkaline protease from pulp and peel of sunflower

Objective: The activity of enzymes participating in the systems of antioxidant protection was assayed in the peel and pulp of sunflower. The essential roles of proteases in food stimulate research to find other sources of the enzyme especially from non-conventional sources. In the present work, we study several biochemical parameters in the pulp and peel of sunflower. Methods: Pulp and peel of sunflower was extracted, antioxidant enzymes and non-enzymatic antioxidant were measured. Alkaline protease was measured and purified from pulp in sunflower. Results: High carbohydrate concentration, beta-carotene, catalase and ascorbate perox-idase activities, free radical scavenging capacity and free flavonoid content were observed in the peel of sunflower. Whereas, MDA and ceruloplasmin activities were high in the pulp of sunflower. Conclusions: The present study concluded that peel in sunflower are strong radical scavengers and can be considered as good sources of natural antioxidants for medicinal and commercial uses. Further analysis showed that protease activity was a significantly high in the pulp compared to the peel.

Alkaline protease from a new halotolerant alkaliphilic Bacillus agaradhaerens strain ak-r isolated from egyptian soda lakes / Protease alcalina de uma nova estirpe ak-r halotolerante e alcalófila de Bacillus agaradhaerens isolada a partir dos lagos alcalinos egípcios

Alkaline proteases are hydrolytic enzymes that cleave peptide bonds in proteins and peptides in alkaline conditions, which occupy a pivotal importance with respect to their industrial applications. This study aimed to isolate new alkaline protease producing alkaliphilic bacteria from Egyptian soda lakes and optimize the fermentation process to enhance the enzyme production. The extensive screening process of the samples collected from Egyptian soda lakes resulted in isolation of a potent alkaline protease producing alkaliphilic strain AK-R. The isolate was identified as Bacillus agaradhaerens strain AK-R based on 16S rRNA gene analysis (99%). Wheat bran and gelatin supported maximum alkaline protease production as carbon and nitrogen sources, respectively. Strain AK-R is halo-tolerant thermotolerant alkaliphilic bacterium in nature, as it can grow over a wide range of NaCl concentrations (up to 25%) and up to 55 °C, with maximal growth and enzyme production at 2.5-5%, and pH 11 at 35 °C. Among the tested cations, only Mg2+ and Ca2+ ions significantly enhanced the enzyme production by about 1.2, and 1.3 fold compared to control, respectively. Alkaline protease secretion was coherent with the growth pattern, reaching maximal yield after about 32 h (mid stationary phase). In conclusion a new halo-tolerant thermo-tolerant alkaliphilic alkaline protease producing Bacillus agaradhaerens strain AK-R was isolated from Egyptian soda lakes. Optimization of the nutritional and cultivation conditions resulted in increase of enzyme yield by 20 fold. Strain AK-R and its extracellular alkaline protease with salt, pH and temperature, tolerance signify their potential application in laundry and pharmaceuticals industries.

Proteases alcalinas são enzimas hidrolíticas que quebram ligações peptídicas em proteínas e peptídeos em condições alcalinas, o que ocupa uma importância fundamental em relação às suas aplicações industriais. Este estudo teve como objetivo isolar novas proteases alcalinas e produzir bactérias alcalófilas a partir dos lagos salgados alcalinos egípcios e otimizar o processo de fermentação para aumentar a produção de enzimas. O extensivo processo de triagem das amostras coletadas dos lagos salgados alcalinos egípcios resultou no isolamento de uma protease alcalina potente produzindo uma estirpe alcalófila AK-R. O isolado foi identificado como sendo a estirpe AK-R de Bacillus agaradhaerens baseado na análise de genes 16S rRNA (99%). O farelo de trigo e a gelatina suportaram a produção máxima de protease alcalina como fontes de carbono e nitrogênio, respectivamente. A estirpe AK-R é uma bactéria alcalófila halotolerante e termotolerante, pois pode crescer dentro de uma vasta gama de concentrações de NaCl (até 25%) e até 55ºC, com crescimento e produção de enzimas máximos a 2.5-5% e pH 11 a 35ºC. Dentre os cátions testados, somente os íons Mg2+ e Ca2+ aumentaram significativamente a produção de enzimas em cerca de 1.2 e 1.3 em comparação ao controle, respectivamente. A secreção de protease alcalina foi coerente com o padrão de crescimento, atingindo o rendimento máximo após 32h (fase estacionária média). Pode-se concluir que uma nova estirpe AK-R de Bacillus agaradhaerens halotolerante, termotolerante e alcalófila produtora de protease alcalina foi isolada a partir dos lagos salgados alcalinos egípcios. A otimização das condições de nutrição e cultivo resultou num aumento da produção de enzima em 20 vezes. A estirpe AK-R e a sua protease alcalina extracelular com tolerância ao sal, pH e temperatura tornam significantes as suas potenciais aplicações nas indústrias farmacêutica e de lavanderia.

One Factor at A Time based optimization of protease from poultry associated Bacillus licheniformis.

Bacteria were isolated from poultry farm of Guduvanchery, Tamil Nadu, India and exhibited variable protease activity on skim milk agar plates. Of 10 bacterial isolates screened, Bacillus licheniformis strain 018 was observed as a hyperprotease producer and it was further characterized using biochemical and molecular tools. Protease production from the isolate was enhanced by optimizing the culture conditions using One Factor at A Time (OFAT) method. The bacteria exhibited its optimal enzyme activity at pH- 9.0, temperature- 35⁰C, agitation speed- 130 rpm, incubation time- 24 h, carbon source- casein and nitrogen source- yeast extract. On the other hand, the crude proteases were found to be significantly active and stable at broad range of pH (5.0-9.0) and temperature (30-60⁰C). To the best of my knowledge this is the first report on the production and enhancement of alkaline protease from poultry farm isolate using OFAT method. Stability of the enzyme at high temperature and pH can be explored for varied industrial applications.

Expression, purification and detection of activities of AprA and its inhibitor AprI from Pseudomonas aeruginosa / 军事医学

Objective To clone and express the alkaline protease AprA , one important virulence factor secreted by Pseudomonas aeruginosa(PAE)in Escherichia coli, to clone and express the inhibitor of AprA (AprI) and its substrate flagellin , and to detect the function of AprA and the inhibitory function of AprI .Methods The genes encoding AprA ,AprI and flagellin gene were amplified respectively by PCR using PAE PAO 1 genome DNA as the template .The expression vec-tors (pET-28a-AprA, pET-28a-AprI and pET-28a-Flagellin) were constructed and transformed into E.coli BL21(DE3) respectively.The recombinant AprA protein was expressed by IPTG induction and purified via denaturing and renaturation. The recombinant AprI and flagellin were expressed and purified by Ni 2+affinity chromatography .The cleavage activities of AprA on flagellin were detected by SDS-PAGE.Results Recombinant AprA , AprI and flagellin protein were expressed and purified .It was demonstrated that AprA cleaved flagellin , which was blocked by AprI .Conclusion Recombinant AprA could cleave its substrates as an alkaline protease , and its inhibitor AprI inhibits the activities of AprA .This study will contribute to further investigations on the role of AprA in the pathogenesis of PAE .

Preparation of anticoagulant peptide from casein and study on thrombolysis and anticoagulation in vitro / 中草药

Objective: To study the preparation and separation method for anticoagulant peptide and the effect of anticoagulation and thrombolysis in vitro. Methods: The casein was hydrolyzed to prepare anticoagulant peptide using the mixed four enzymes such as papain, pineapple proteinase, neutral protease, and alkali protease. The anticoagulant peptide was extracted using immobilized thrombin. The effect of haemolysis and anticoagulation in vitro was investigated through the New Zealand rabbits experiments. Results: The conditions of preparation anticoagulant peptide were as follows: quality of casein was 15%, papain proteinase, pineapple proteinase, neutral protease, and alcalase dosage were 1500, 2400, 1000, and 1250 U/(g casein), respectively, temperature was 50℃, pH value was 7.0, and hydrolysis time was 4 h. The conditions for the extraction of anticoagulant peptide were as follows: the initial concentration was 6 ATU (Anti Thrombin Unit)/mL, temperature was 30℃, pH value was 5.0, and time was 30 min. Anti-extraction temperature was 30℃, pH value was 7.6, and time was 40 min. The purified anticoagulant peptide was analyzed via high performance size exclusion chromatography. The molecular weight of purified anticoagulant peptide was equal to N-Hippuryl-His-Leu hydrate and the main components were three peptides. The time of anticoagulation was more than 72 h and the time of hemolysis was 24 h in vitro. Conclusion: The main components of anticoagulant peptides are three peptides. The effect of hemolysis and anticoagulation in vitro is good.

Electrophoresis separation and MS analysis of proteins in fermentation broth of Poria cocos / 中草药

Objective: In order to carry out the study on proteins from Poria cocos fermentation broth, the proteins in the fermentation broth were separated and identified. Methods: Proteins were obtained by organic acid precipitation from the fermentation broth and the protein concentration was determined by Bradford method. The obtained P. cocos secreted proteins were separated on SDS-PAGE electrophoresis, subjected to in-gel digestion, then identified by mass spectrometric analysis followed by database searching. Results: The protein concentration in the fermentation broth was around 74.01 μg/mL, with the apparent molecular weight ranged from 2.8 × 104 to 1.3 × 105. A total of 52 P. cocos secreted proteins were identified, including catalase, protein kinase, alkaline protease, glucoamylase, lysozyme, and so on. Conclusion: P. cocos fermentation broth has abundant proteins, which could be a good material for the study of P. cocos protein and also a potential healthy food and beverage.

Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

Background Alkaline proteases are among the most important classes of industrial hydrolytic enzymes. The industrial demand for alkaline proteases with favorable properties continues to enhance the search for new enzymes. The present study focused on isolation of new alkaline producing alkaliphilic bacteria from hyper saline soda lakes and optimization of the enzyme production. Results A new potent alkaline protease producing halotolerant alkaliphilic isolate NPST-AK15 was isolated from hyper saline soda lakes, which affiliated to Bacillus sp. based on 16S rRNA gene analysis. Organic nitrogen supported enzyme production showing maximum yield using yeast extract, and as a carbon source, fructose gave maximum protease production. NPST-AK15 can grow over a broad range of NaCl concentrations (0-20%), showing maximal growth and enzyme production at 0-5%, indicated the halotolerant nature of this bacterium. Ba and Ca enhanced enzyme production by 1.6 and 1.3 fold respectively. The optimum temperature and pH for both enzyme production and cell growth were at 40°C and pH 11, respectively. Alkaline protease secretion was coherent with the growth pattern, started at beginning of the exponential phase and reached maximal in mid stationary phase (36 h). Conclusions A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.

Production and Partial Characterization of Alkaline Protease from Bacillus Tequilensis Strains CSGAB0139 Isolated from Spoilt Cottage Cheese.

An alkaline protease producing strain was isolated from spoilt cottage cheese sample which was identified as Bacillus tequilensis strain SCSGAB0139 on the basis of morphological, cultural, biochemical characteristics and 16S rRNA sequence analysis. Primary screening for protease production was carried out by observing for zone of hydrolysis on skim milk agar, GYEA milk agar and gelatin agar plates. Physicochemical parameters like pH of the medium, incubation time and temperature, aeration and composition of the medium were optimized for maximum protease production by this isolate. Maximum yield of protease (85.67U/ml) was obtained in a medium containing peptone (5% w/v), maltose (5% w/v) and KNO3, 0.5%; K2HPO4, 0.4%; trisodium citrate, 0.4; CaCl2, 0.0002%; MgSO4·7H2O, 0.05%; Na2CO3, 1%.; 1% (v/v) of a trace element solution (NH4)6MO7O24, 0.01%; FeSO4·7H2O, 0.2%; CuSO4·5H2O, 0.02%; ZnCl2, 0.02%) having pH 10, inoculated with 1%(v/v) of pre-grown cell mass and incubated at 30°C on a rotary shaker (100rpm) for 48hrs. Absence of any one of the following salts viz. KNO3, K2HPO4, tri-sodium citrate; MgSO4, CaCl2 and Na2CO3 from optimized medium reduced the protease production by 80% to 40%. The enzyme has an optimum pH of 9 and maintained its stability over a broad pH range between 6 and 10. Its optimum temperature is 30°C, and exhibited a stability of up to 65°C. Among metal ions only Ca2+ and Mg2+ions enhanced the enzyme activity up to 105% and 107% respectively while other metal ions reduced the activity by 40% where as EDTA exhibited the least inhibitory effect upon the enzyme. Protease activity was enhanced in the presence of isopropanol and marginally reduced in the presence of other organic solvents studied. The crude enzyme showed stability towards various surfactants such as Tween-20, Tween- 80, SDS and Triton X-100. It also showed excellent stability and compatibility with commonly used laundry detergents (Ariel, Surf excel and Surf Blue). The enzyme retained its activity in 2% H2O2 indicating it to be bleach-stable. The present findings show that the protease from Bacillus tequilensis strain SCSGAB0139 is alkali- and detergent- stable and hence, when this protease was applied to remove blood stains from cotton fabrics indicated its potential use in detergent formulations.

Partial purification and characterization of extracellular protease from a halophilic and thermotolerant strain Streptomyces pseudogrisiolus NRC-15.

An alkaline protease was purified from a halophilic and thermotolerant potent alkaline protease-producing strain Streptomyces pseudogrisiolus NRC-15 using ammonium sulphate precipitation and Sephadex G-100 column chromatography. The enzyme was purified to 77.24-folds with a yield of 91.8% and the specific activity was 112 U/mg of protein. The protease showed a single band on SDS-PAGE with its molecular mass at 20 kDa and exhibited a maximum relative activity of 100% using casein as a substrate and. The enzyme had an optimum pH of 9.5 and displayed optimum activity at 50°C. The enzyme activity was completely inhibited by the serine protease inhibitor PMSF, suggesting the presence of serine residue in the active site. The enzyme activity was increased by the metal ions Ca2+, Co2+, K+ and Mg2+. The enzyme significantly enhanced the removal of stains when used with wheel detergent, indicating the potential of the enzyme for using as a laundry detergent additive to improve the performance of heavy-duty laundry detergent.

Purification and characterization of an extracellular alkaline protease produced from an isolated bacillus subtilis.

This paper describes the studies on the purification and partial characterization of serine alkaline protease produced through submerged fermentation process from a locally isolated Bacillus subtilis. This strain, grown in a highly alkaline medium (pH 10), produces an extracellular proteolytic enzyme. The alkaline protease was purified in a simple two-step procedure involving ammonium sulphate precipitation and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified alkaline protease indicated an estimated molecular mass of 30KDa. It was more active in the range of 20-60ºC and had an optimum activity at 55ºC with optimum pH of 10.5. Characterization of the protease showed that it required certain cations such as Mg++, Mn++ and Ca++ for maximal activity. The serine nature of the alkaline protease was confirmed by PMSF inhibition. The temperature and pH stability of this Alkaline Protease from Bacillus Subtilismakes it potentially useful forindustrial applications.

Alkaline protease production, extraction and characterization from alkaliphilic Bacillus licheniformis KBDL4: A Lonar soda lake isolate.

A bacterium producing an alkaline protease was isolated from the Lonar soda lake, Buldhana district (19°58' N; 76°31' E), Maharashtra, India. The most appropriate medium for the growth and protease production was composed of (g/L): casein 10; yeast extract 4; KH2PO4 0.5, K2HPO4 0.5 and CaCl2 0.5. The enzyme showed maximum activity with and without 5 mM Ca2+ at 70 and 60 oC, respectively. The enzyme retained 40 and 82% of its initial activity after heating for 60 min at 60 oC, in absence and presence of 5 mM CaCl2 respectively. The enzyme remained active and stable at pH 8-12, with an optimum at pH 10. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. It also showed excellent stability and compatibility with commonly used laundry detergents. Wash performance analysis revealed that enzyme could effectively remove blood stains. It also showed decomposition of gelatinous coating on X- ray film.

Studies on the Catalytic Properties of Partially Purified Alkaline Proteases from Some Selected Microorganisms

Aims: The research was done to study the conditions enhancing catalytic activities of alkaline proteases from Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp., Corynebacterium sp. and Bacillus subtilis. Methodology and Results: The proteolytic enzymes were purified in 2-step procedures involving ammonium sulphate precipitation and sephadex G-150 gel permeation chromatography. The upper and lower limits for the specific activities of proteases from the selected microorganisms were estimated at 20.63 and 47.51 units/mg protein with Zymomonas protease having the highest specific activity towards casein as its substrate and purification fold of 3.46, while that of Lactobacillus brevis protease was 8.06. The native molecular weights of these active proteins ranged from 30.4 to 45.7 kDa with Athrobacter sp. protease having the highest weight for its subunits. The proteolytic enzymes had optimum pH range of 8 to 10 and temperature range of 50 to 62 ºC accounting for the percentage relative activity range of 75 to 94% and 71 to 84 % respectively. The activities of Lactobacillus brevis and Bacillus subtilis proteases were maximum at pH 9 and 10 respectively. Lactobacillus brevis protease activity was maximum at temperature of 62 ºC, while beyond this value, a general thermal instability of these active proteins was observed. At above 70 ºC, the catalytic activities of Corynebacterium sp., Vibrio sp., Zymomonas sp. and Arthrobacter sp. proteases were progressively reduced over a period of 120 min of incubation, while Bacillus subtlis and Lactobacillus brevis proteases were relatively stable. Effect of metal ions was investigated on the catalytic activity of protease from the microorganisms. Lactobacillus brevis, Zymomonas sp., Arthrobacter sp., Corynebacterium sp. and Bacillus subtilis protease activities were strongly activated by metal ions such as Ca+2 and Mg+2. Enzyme activities were inhibited strongly by Cu2+ and Hg2+ but were not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with K+, Na+ and Fe2+. Conclusion, significance and impact of study: The outcome of this present study indicated useful physico-chemical properties of proteolytic enzymes that could be of biotechnological use in enhancing enzyme catalytic efficiency.

Characterization of protease from Alcaligens faecalis and its antibacterial activity on fish pathogens.

Alcaligens faecalis AU01 isolated from seafood industry effluent produced an alkaline protease. The optimum culture conditions for growth as well as enzyme production were 37oC and pH 8. The partially purified protease had specific activity of 9.66 with 17.77% recovery with the molecular weight of 33 kDa and it was active between 30-70oC and optimum being at 55oC and pH 9. The enzyme retains more than 85% activity at 70oC and 78% even at pH 10.The enzyme inhibited the growth of fish pathogens such as Flavobacterium sp., Pseudomonas fluorescens, Vibrio harveyi, Proteus sp. and Vibrio parahaemolyticus. From the present study it can be concluded that Alcaligens faecalis AU01 has the potential for aquaculture as probiotic agent and other several applications.

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40°C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu­­­2+, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing

The purpose of the present investigations was to improve the yield of alkaline protease for leather dehairing by subjecting the indigenous proteolytic strain Bacillus licheniformis N-2 to various mutagenic treatments viz. UV irradiations, NTG (N-methyl-N-nitro-N-nitrosoguinidine) and MMS (methyl methane sulfonate). After screening on skim milk agar plates, a total of nine positive mutants were selected for shake flask experiments. Among these, the best proteolytic mutant designated as UV-9 showed 1.4 fold higher alkaline protease activity in preoptimized growth medium than the parent strain. The fermentation profile and kinetic parameters such u(h-1), Yp/s, Yp/x, Yx/s, q s, Qs, q p and Qp also indicated the superiority of the selected mutant UV-9 for alkaline protease production over the parent strain and rest of the mutants. The dehairing capability of mutant UV-9 alkaline protease was analyzed by soaking goat skin pieces for different time intervals (3-15 h) at 40 º C. A complete dehairing without degradation of collagen was achieved after 12 h, indicating its commercial exploitation in leather industry.