Maximum Protease Production Research Articles

Production and optimization of thermophilic alkaline protease in solid-state fermentation by Streptomyces sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g(-1)) when compared to individual WB (74.50 U g(-1)) or CDS (69.50 U g(-1)) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g(-1)) was obtained with an initial moisture content of 60%, an inoculum level of 1 x 10(8) (spore g(-1) substrate) when incubated at 45 degrees C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g(-1) under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.

Characterization of Chryseobacterium aquaticum Strain PUPC1 Producing a Novel Antifungal Protease from Rice Rhizosphere Soil

Strain PUPC1 produces an antifungal protease as well as plant growth promoting enzymes such as 1- aminocyclopropane-1-carboxylate (ACC) deaminase and phosphatase. Morphological, cultural, and physiological characteristics as well as 16S rRNA gene-sequence-based phylogenetic analysis confirmed the taxonomic affiliation of PUPC1 as Chryseobacterium aquaticum. The optimum growth of PUPC1 was observed at pH 6.0 and 30 degrees , and maximum protease production was observed in medium B amended with 1% tryptone, 0.5% sucrose, and 0.005% MnCl2. The protease was purified by ammonium sulfate precipitation, Sephadex G-75 gel filtration chromatography, and electroelution from preparative SDS-PAGE. The protease had a molecular mass of 18.5 kDa. The optimum pH and temperature stability of the protease were pH 5.0- 10.0 and temperature 40-70 degrees . Chryseobacterium aquaticum PUPC1 and its protease showed a broad-spectrum antifungal activity against phytopathogenic fungi. Strain PUPC1 also exhibited plant growth promoting traits. The objective of the present investigation was to isolate a strain for agricultural application for plant growth promotion and biocontrol of fungal diseases.

Production of alkaline protease from Cellulosimicrobium cellulans

Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC.

Immobilization of Bacillus circulans MTCC 7906 for enhanced production of alkaline protease under batch and packed bed fermentation conditions

Alkaline protease production by immobilized cells of Bacillus circulans MTCC 7906 was studied using agar-agar and calcium alginate as entrapment materials. Under batch conditions using agar agar, 2% agar concentration, 6.25mm bead size and 200 beads/ flask inoculum size were the optimal conditions for a maximum alkaline protease production of 19.9 IU/ml and while for alginate beads, 3% alginate concentration, 5.2mm bead size and 200 beads/ flask inoculum size were the optimal conditions for a maximum alkaline protease production of 19.7 IU/ml. The alginate and agar agar beads were reusable for four and nine successive batch cycles respectively. A maximum enzyme production of 28 and 27 IU/ml was obtained with incubation time of 36 h, column length of 30cm and flow rate of 10ml/h with agar-agar and alginate entrapped cells respectively under packed bed fermentation conditions. Scanning electron microscopy (SEM) of the agar agar entrapped cells showed a random distribution of cells on the surface with pockets of high cell density.

Some properties of extracellular protease from Bacillus licheniformis LBBL-11 isolated from iru, a traditionally fermented African locust bean condiment

Twelve strains of Bacillus licheniformis isolated from traditionally fermented African locust bean (iru) were evaluated in respect to production of protease on skim milk agar. B. licheniformis LBBL-11 exhibited the highest proteolytic activity with a diameter of clear zone measuring 35.0 mm. Production of protease from B. licheniformis LBBL-11 was further studied by growing the strain on nutrient broth. Maximum protease production was 18.4 U/ml at 48 hour of growth which coincided with the end of exponential phase. The protease from this Bacillus sphad optimum pH of 8.0 and was stable over a wide pH range of 5.0 - 11.0. The optimum temperature for the protease activity was 60oC. The enzyme was 95% stable at 60oC after 60 min of incubation. These properties indicate possible application of B. licheniformis LBBL-11 as potential starter culture for the fermentation of African locust bean under controlled conditions of temperature and pH. Key words: Fermented locust bean, iru, protease, thermostable, Bacillus licheniformis

CHARACTERIZATION OF HALOPHILIC BACTERIUM NB2-1 FROMPLA-RAAND ITS PROTEASE PRODUCTION

ABSTRACT A halophilic proteolytic bacterium, NB2-1, isolated from Pla-ra, fermented fish in Thailand, was identified as Virgibacillus marismortui on the basis of the genetic and phenotypic characteristics. NB2-1 produced a substantial level of extracellular proteolytic activity, corresponding with the growth and reaching the maximum at the end of the exponential phase (3 days). The optimum conditions required for maximum protease production were 15% (w/v) NaCl, pH at 9 and temperature at 30C. Of the media tested, maximum protease production occurred in a medium containing 15% NaCl, 0.5% yeast extract, 0.1% glutamic acid, 0.2% KCl, 0.3% trisodium citrate, 2% MgSO4·7H2O, 0.0036% FeCl2·4H2O and 0.000036% MnCl2·4H2O. The protease activity was optimally active at pH 10, 50C and 5% (w/v) NaCl when casein was used as a substrate. The activity was strongly inhibited by phenylmethane sulfonyl fluoride. Activity-stained substrate gel indicated the presence of several proteases with the estimated molecular weights of 17, 19, 24, 29 and 35 kDa in the crude NB2-1 extracellular protease. The results of the present investigation showed that the strain NB2-1 could be a potential source of thermoactive alkaline protease production. PRACTICAL APPLICATIONS Halophiles are the most likely source of enzymes that not only are salt-tolerant but also thermotolerant. They constitute a heterogeneous group of microorganisms including species belonging to different genera. The isolation of halophiles able to produce extracellular enzymes will provide the possibility to have optimal activities at different salt concentrations. Virgibacillus marismortui NB2-1, a moderately halophilic bacterium isolated from Pla-ra, produced a substantial level of extracellular proteolytic activity that was active in extreme conditions. Using casein as a substrate, the protease exhibited maximal activity at pH 10, 50C in the presence of 5% (w/v) NaCl. The results of the present investigation showed that the strain NB2-1 could be a potential source of thermoactive alkaline protease production.

Evaluation of Protease-producing Ability of Fish Gut Isolate Bacillus cereus for Aqua Feed

Extracellular protease production by Bacillus cereus isolated from the intestine of fish Mugil cephalus has been investigated in shake-flask experiment using different preparations of tuna-processing waste such as raw fish meat, defatted fish meat, alkali hydrolysate, and acid hydrolysate as nitrogen source. Among the tuna preparations tested, defatted fish meat supported the maximum protease production (134.57 ± 0.47 U ml−1), and 3% concentration of the same was found to be optimum for maximizing the protease production (178.50 ± 0.28 U ml−1). Effect of carbon sources on protease production in the optimized concentration of defatted tuna fish meat revealed that galactose aided the higher protease production (259.83 ± 0.04 U ml–1) than the other tested carbon sources and a concentration of 1.5% galactose registered as optimum to enhance the protease production (289.40 ± 0.16 U ml−1). The halotolerancy of B. cereus for protease production indicated that 3% of sodium chloride was optimum to yield maximum protease (301.63 ± 0.20 U ml−1). Among the surfactants tested, protease production was high in Triton X 100-added medium (298.63 ± 0.12 U ml−1) when compared to other surfactants, and its optimum concentration recorded was 0.8% (320.57 ± 0.17 U ml−1) for more protease production. Partial characterization of crude enzyme revealed that pH 7.0 (278.90 ± 0.08 U ml−1) and 60°C temperature (332.37 ± 0.18 U ml−1) were optimum for better protease activity by B. cereus.

Production of moderately halotolerant, SDS stable alkaline protease from Bacillus cereus MTCC 6840 isolated from lake Nainital, Uttaranchal state, India

A moderately cold active, extracellular alkaline protease producing bacterium was isolated from a fresh water lake. The isolate was found to be a gram-positive, rod shaped organism later identified as Bacillus cereus MTCC 6840. The bacterium produced the maximum amount of enzyme when allowed to grow for 24 h at temperature 25º and pH 9.0. Among a variety of substrates used, fructose as a carbon source and a combination of yeast extract and peptone as nitrogen source, supported the maximum protease production by the organism (120 U/ml). Fe++ and Co++ stimulated the enzyme activity whereas Ca++, Cu++, K+, Mg++ and Mn++ inhibited it to different extents. The protease was found to be highly stable in the presence of NaCl, SDS and acetone. Treatment with EDTA and PMSF resulted in the considerable loss of enzyme activity. The enzyme was found to be optimally active at pH 9.0 and temperature 20ºC.

Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrica grass and potato peel as low-cost medium: Characterization and application of enzyme in detergent formulation

Among the different agro-industrial waste products and kitchen waste materials, viz. mustered oil cake, wheat bran, rice bran, Imperata cylindrica grass, banana leaves, potato peels and used tea leaves screened as substrates/solid supports for the production of alkaline protease by a thermophilic strain of Bacillus subtilis DM-04 under solid-state fermentation, potato peel followed by I. cylindrica grass supported maximum protease production. Further, potato peel and I. cylindrica grass mixed in a ratio of 1:1 (w/w) significantly ( P < 0.05) enhanced the protease production by B. subtilis DM-04 compared to individual substrate. Among the tested nitrogen compounds, 0.1% (w/w) beef extract followed by yeast extract served as the best co-nitrogen sources for protease production by B. subtilis DM-04 strain on I. cyindrica grass. Distilled water adjusted to pH 8.0 was most efficient moistening agent for protease production. The crude protease displayed optimum activity at 37–45 °C temperature range and showed characteristics pH optima at pH 8.0–9.0. The protease from B. subtilis DM-04 retained 67% of its original activity post-heating at 60 °C for 15 min. Among the tested protein substrates, casein served as the most preferred substrate for the enzyme. Crude protease exhibited a significant stability and compatibility with most of the tested commercial laundry detergents, demonstrating its feasibility for inclusion in laundry detergent formulation.

Response surface optimization of medium composition for alkaline protease production by Bacillus clausii

A culture medium for production of bacterial alkaline protease was developed following preliminary studies by means of response surface method. Central composite design was applied to optimize the medium constituents and explain the combined effects of three medium constituents, viz., sucrose, yeast extract and potassium nitrate. The optimum values for the tested variables were; sucrose 11 g l −1, yeast extract 5 g l −1 and KNO 3 5.2 g l −1. A second-order model equation was suggested and then validated experimentally. The model adequacy was very satisfactory as the coefficient of determination was 0.94. The maximum alkaline protease production was 1520 ± 35 U ml −1at 42 h of incubation which showed about 1.5-fold increase in protease production over the central point and an overall 6-fold enhancement (1520–250 U ml −1) over the basal medium.

Enhanced production and characterization of a solvent stable protease from solvent tolerant Pseudomonas aeruginosa PseA

Enzymes stable and active in non-aqueous media are in large demand for their increasing application in organic synthesis. A solvent-stable protease is reported from a solvent tolerant strain (PseA) of Pseudomonas aeruginosa. Production of this novel protease was optimized by varying the culture conditions. Glycerol (0.7%) as carbon source, casein (0.4%) and yeast extract (0.6%) as nitrogen sources, CaCl 2 (0.6 mM) as metal ion in culture media (pH 7.0), inoculated with 2.5% of 36 h grown mother culture and incubation for 24 h at 30 °C with 250 rpm shaking were found to be the best culture conditions leading to maximum growth and protease production (1601 U/ml). The enzymatic properties of the protease revealed, pH and temperature optimum of 9.0 and 55 °C, respectively. It retained 100%, 91% and 80% of its initial activity after heating for 30 min at 45, 50 and 55 °C, respectively. The enzyme exhibited remarkable stability towards range of hydrophobic organic solvents. This unique property makes it attractive for use in non-aqueous enzymology.

Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods

Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2 4 full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett–Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl 2, 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.

Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate

Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO 3 for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production.

Optimization of cold-active protease production by the psychrophilic bacterium Colwellia sp. NJ341 with response surface methodology

Culture conditions were optimized for an extracellular cold-active protease production by the psychrophilic bacterium Colwellia sp. NJ341. Response surface methodology was applied for the most significant fermentation parameters (casein, citrate sodium, temperature and Tween-80) identified earlier by one-factor-at-a-time approach. A 2 4 full factorial central composite design was employed to determine the maximum protease production. Using this methodology, the quadratic regression model of producing cold-active protease was built and the optimal combinations of media constituents for maximum protease production (183.21 U/mL) were determined as casein 5.18 g/L, citrate sodium 3.84 g/L, temperature 7.96 °C, Tween-80 0.23 g/L. Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate.

Alkaline protease production by submerged fermentation in stirred tank reactor using Bacillus licheniformis NCIM-2042: Effect of aeration and agitation regimes

The effects of aeration and agitation on alkaline protease production and protease yield were studied by submerged fermentation in a batch STR using Bacillus licheniformis NCIM-2042. The agitation rate varied in the range of 200–400 rpm at each airflow rates of 1–3 vvm. The maximum protease production was found on third day (72 h) and then decreased during fourth and fifth day of operation in all batches of STR operation. The best combination of airflow and agitation rate for the present system was at 3 vvm of airflow rate and 200 rpm of agitation rate on the basis of maximum specific protease production rate. Maximum specific protease production of 102 U/mg DC was observed on third day at 3 vvm and 200 rpm. The effects of casein concentrations on protease production, viscosity changes and oxygen transfer rate were studied. The increase in casein concentration has resulted in increased viscosity of the fermentation broth with increase in time of bioreactor operation, which in turn resulted in decreased oxygen mass transfer coefficient with reduced oxygen transfer rates.

Alkaline protease production by a strain of marine yeasts

Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45°C. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5g soluble starch and 2.0g NaNO3 in 100mL seawater with initial pH 6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5°C, aeration rate 8.0L min−1 and agitation speed 150r min−1 Under the optimal conditions, 623.1 U mg−1 protein of alkaline protease was reached in the culture within 30h of fermentation.

Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326

Bacillus cereus MCM B-326, isolated from buffalo hide, produced an extracellular protease. Maximum protease production occurred (126.87 ± 1.32 U ml −1) in starch soybean meal medium of pH 9.0, at 30 °C, under shake culture condition, with 2.8 × 10 8 cells ml −1 as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25–65 °C and pH 6–12, with maximum activity at 55 °C and pH 9.0. The enzyme required Ca 2+ ions for its production but not for activity and/or stability. The partially purified enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be effectively used to remove hair from buffalo hide indicating its potential in leather processing industry.

Influence of media composition on acid protease production by &lt;i&gt;Aspergillus niger&lt;/i&gt; (NRRL 1785)

Aspergillus niger (NRRL 1785) has been reported as a good producer of acid protease with optimum pH of 4.0 using glucose as the sole carbon source and peptone as sole nitrogen source. The production of acid protease by A.niger (NRRL 1785) using other carbon sources (lactose, sucrose, starch, maltose and glycerol) to substitute for glucose and other nitrogen sources (urea, casein, beef extract, groundnut meal and corn steep liquor) to substitute for peptone were studied. The effects of media composition on acid protease production were determined. All the fermentation media with different carbon and nitrogen sources tested supported acid protease production. Peak of enzyme production for all media composition was at the 96th hour of fermentation though at varying yields. Lactose supported the maximum production of 23.80 UmIi-1 amongst the tested carbon source (glucose substitutes) while beef extract supported maximum protease production amongst peptone substitutes with protease activity of 19.95 UmI-1. None of the media tested measured up to the protease activity of 26.05 UmI-1. The result of this study gives a strong indication that A.niger (NRRL 1785) is capable of utilizing different carbon and nitrogen sources for acid protease production. Economically, this strain seems to be promising as an alternative microorganism for acid protease production. Keywords: acid protease, Aspergillus niger (NRRL 1785), fermentation media Global Journal of Pure and Applied Sciences Vol. 12(1) 2006: 85-88

Purification and characterization of an extracellular haloalkaline protease produced by the moderately halophilic bacterium, Salinivibrio sp. strain AF-2004

Among several strains of moderately halophilic bacteria that were isolated from various areas in Iran, strain AF-2004 was selected as the best producer of extracellular protease and was used for further studies. Phenotypic classification and 16S rRNA sequence analysis placed AF-2004 in the genus Salinivibrio. Maximal protease production was detected at the end of exponential growth phase in the medium containing 5% (w/v) NaCl. This protease was purified to homogeneity by a combination of acetone precipitation, Q-Sepharose ion exchange and Sephacryl S-200 gel filtration chromatography. The enzyme was a monomeric protease with a relative molecular mass of 38–43 kDa by SDS-PAGE and gel filtration chromatography. The specific activity of the purified enzyme was determined to be 171.6 μmol of tyrosine/min per mg of protein using casein as a substrate. The enzyme exhibited its optimal activity at 65 °C, pH 8.5, and 0–0.5 M NaCl with a high tolerance to salt concentrations of up to 4 M. The protease was identified as a zinc-metalloprotease, which was strongly inhibited by EDTA and 1,10-phenanthroline, and the N-terminal amino acid sequence determined showed high similarity to the zinc-metalloproteases from Vibrio species.

Green gram husk—an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation

Alkaline protease production under solid-state fermentation was investigated using isolated alkalophilic Bacillus sp. Among all agro-industrial waste material evaluated, green gram husk supported maximum protease production. Solid material particle size regulated the enzyme production and yield was improved with the supplementation of carbon and nitrogen sources to the solid medium. Optimum enzyme production was achieved with 1.5% maltose and 2.0% yeast extract with 371% increase than control. Glucose did not repressed enzyme production but inorganic nitrogen sources showed little negative impact. The physiological fermentation factors such as pH of the medium (pH 9.0), moisture content (140%), incubation time (60 h) and inoculum level played a vital role in alkaline protease production. The enzyme production was found to be associated with the growth of the bacterial culture.