Isolation, Purification, and Optimization of Thermophilic and Alkaliphilc Protease Originating from Hot Water Spring Bacteria (original) (raw)
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Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101
Applied Microbiology and Biotechnology, 1989
A thermophilic strain producing thermostable protease and cellulase was isolated from hot water springs. The strain was identified as Bacillus licheniformis on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was partially purified by ammonium sulphate precipitation. Thermostable alkaline protease showed highest activity and stability at pH (10-11) and at temperature 55°C. Enzyme activity increased in the presence of metal ions like Ca 2+ and Cu 2+. The bacterial isolate produces a serine protease. The supplementation of the enzyme in detergents could significantly improve the cleansing performance towards the blood stains and suggest its possible use as a detergent additive. The enzyme also showed potential for application as a dehairing agent.
This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214 U/ml) under optimized conditions: carbon source, citric acid-1.5 % (w/w); inducer, soyabean meal-2 % (w/w); pH 11.0; shaking condition 37°C for 48 h. The enzyme had pH and temperature optima of 9.0 and 60°C, respectively. The enzyme displayed the molecular mass of 36 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis study and exhibited activity at a wide range of pH (6.0-11.0) and thermostability (20-70°C). More than 70 % residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H 2 O 2 for 30 min. The protease activity was also enhanced by divalent cations such as Ba 2+ , Ca 2+ and Mg 2+ and was strongly inhibited by Fe 2+ , Zn 2+ , Sr 2+ , Hg 2+ and urea. The enzyme retained more than 50 % of its initial activity after pre-incubation for 1 h in the presence of 5 % (v/v) organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1 % w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the ecofriendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications.
A Review on Microbial Alkaline Protease: An Essential Tool for Various Industrial Approaches
Industrial Biotechnology
Proteolytic enzymes are present in all living organisms and help in cell growth and differentiation. Proteases are the hydrolytic enzymes that act as biocatalysts for the cleavage of proteins into smaller peptides and amino acids. Microorganisms have turned out to be a competent and inexpensive source of alkaline protease enzymes that can produce a continuous and consistent supply of desired product. Alkaline proteases have extensive application in various industrial sectors especially in detergent and leather industries. However, their application in food has not been much exploited. This review summarizes all the reports of applications of alkaline protease in different sectors with a main view on food applications. The effect of various physiochemical parameters on alkaline protease is discussed. Different sources of isolation and optimum pH and temperature of alkaline protease producing bacterial and fungal species are also reported.
Pure and Applied Biology
The purpose of this study was production of protease enzyme by isolated bacteria and evaluation of their potential for various industrial applications. Two strains AN16 and AN13 exhibiting maximum proteolytic activity were used for further studies. The activity of crude enzymes was completely inhibited by phenyl-methyl-sulphobnyl fluoride (PMSF) and ethylene diamine tetraacetic acid (EDTA) had no such negative effect on activity. Enzymes were relatively stable in presence of surface active agents such as Triton X-100, Tween-80, Tween-20 and oxidizing agent H₂O₂. The enzymes exhibited maximum activity within pH range of 7-9. The optimum temperature was recorded to be 37⁰C. The relative activity of isolate AN13 protease in presence of metal ions Ca2+,
Molecular Biology Reports, 2012
Proteases have prospective financial and environment-friendly applications; hence attention is focused currently on the finding of new protease producing microorganism so as to meet the requirements of industry. A thermophilic bacterial strain producing extracellular protease activity was isolated from soil and identified as Bacillus cereus by analysis of 16S rRNA. Protease production by the microorganism was improved by studying the impact of the type of nitrogen and carbon source, fermentation period, growth temperature and initial pH of the culture medium in cultivation optimization experiments. The enzyme was purified to homogeneity in two step procedure involving Sephadex G-75 and Q-Sepharose chromatography. The molecular weight of purified enzyme was found to be 58 kDa by SDS-PAGE. Protease exhibited a pH and temperature optima of 7.5 and 60°, respectively. The enzyme was active in the pH range of 6.0-9.0 and stable up to 70°C. Histological analysis of protease treated goat and cow skin pelts showed complete removal of non leather forming structures such as hair shaft, hair follicles and glandular structures. The protease showed the stain removing property from blood stained cotton cloth and found to be compatible with six commercially available detergents. The protease could release peptides from natural proteins after digestion of coagulated egg albumin and blood clot.
Biologia, 2012
An extracellular alkaline protease-producing Vibrio sp. was isolated from mangrove sediments of Vellar estuary. A 9.36-fold purification was achieved by a three-step purification procedure and the molecular weight of the enzyme was determined as 33 kDa by SDS-PAGE. The enzyme was active in a broad range of pH (6.0–11.0) and temperature (30–70°C), the optimum being at pH 9.0 and temperature 55°C. The enzyme was stable at alkaline pH range of 9–11 and up to a temperature of 60°C, after incubation for 1 h. Metals like Co2+, Hg2+, Ni2+ and Cu2+ inhibited the enzyme activity, whereas Fe2+, Ca2+ and Mn2+ were found to enhance the activity. The protease was found to be highly stable in the presence of oxidizing agents like H2O2, detergents such as SDS and Triton-X-100 and also some of the commonly used commercial detergents. The organic solvents like xylene, isopropanol, hexane and benzene were found to enhance as well as stabilize the enzyme activity. The extracellular production of the e...
Environmental pollution caused by chemical based industries has necessitated the development of enzyme based processes as alternatives to the currently employed chemical processes. Bacterial extracellular alkaline proteases are of great importance due to its wide variety of applications in detergent,bioremediation,food,leather processing and pharmaceutical industries. A number of bacterial isolates producing protease were selected from the garden soil and primary screening was achieved by casein hydrolysis method. In them 20 isolates were selected,among them one isolate exhibited the highest proteolytic activity with a clear zone of 25mm. The isolate has been identified by scanning electron microscope, biochemical characterization, 16SrRNAsequencing andphylogenetic analysis as bacillus subtilis.The stability of the enzyme in presence of various detergents were remarkable. The results of the washing performance with detergents were clearly indicated and the removal of blood stains, egg yolk, chocolate stains and dehairing of goat skin suggested the crucial application in the commercial products at large scale and eliminate the use of pollution causing chemicals such as sodium sulphideand lime, greatly helping to prevent environmental pollution.
Bacterial alkaline proteases are of great importance due to its wide spectrum applications in detergent industries, bioremediation, food industries, and leather processing, bio-film degradradation, pharmaceuticals industry, meat tenderizers, protein hydrolyzates, food products and even in the waste processing. Bacillus sp. - the most widely exploited alkaline proteases producer, are often commercially used in bioremediation mixes. In present study 38 isolates were isolated from the soil sample of Kashmir region and screened for protease activity on skim milk agar and casein agar medium. All the isolates showed protease activity at alkaline pH ranging from 9-14 and at low temperature of 0-10ºC. Out of these, MLP1 isolate was further characterized biochemically in order to establish their phylogeny and belongs to Bacillus species. Optimization of pH and temperature conditions for the production of enzyme were determined and found to be 12 and 4 ºC respectively. Future studies involve ...
African Journal of Biotechnology, 2011
Soil samples from different habitats including tanneries, soap industries, garden soil and soil compost were screened for the presence of alkalophilic Bacillus isolates capable of producing alkaline protease in large quantities. One hundred and eighteen (118) isolates were found having proteolytic activity on skim milk agar plates. Isolates forming larger zones, as a result of casein hydrolysis were further studied for quantitative production of extracellular alkaline protease activity in the shake flask studies. Isolate CEMB10370 gave maximum activity. Time course studies indicated that strain CEMB10370 had the highest protease activity (380 APU/mL) after 48 h of fermentation. The wild type enzyme was biochemically characterized. The enzyme exhibits optimal activity at 50°C and pH 11.5. The protease enzyme was completely inhibited by phenylmethylsulfonyl (PMSF, serine protease inhibitor) and its isoelectric point was ~9.5. The enzyme was purified by ion-exchange chromatography using CM-Sepharose column as a ~29 Kilo Dalton (kDa) protein.
Food and Bioproducts Processing, 2014
A marine bacterium Bacillus alveayuensis CAS 5 produced protease when grown at 55 • C for 60 h in 100 ml of basal medium containing 1% SSP (w/v) and purified to 7.77 fold with specific activity of 518.78 U/mg. The optimum temperature, pH and NaCl for enzyme activity were 50 • C, 9 and 35% respectively. The enzyme was highly stable even at 80 • C, pH 12, 35% NaCl and presence of ionic, non-ionic and commercial detergents. The protease was investigated for its application as cleansing additive in blood stain removal. The preset study emphasized that marine wastes can be utilized to generate high value-added products and hidden potential in the production of functional foods.