Maximum Protease Production Research Articles

Production of feather hydrolysates with antioxidant, angiotensin-I converting enzyme- and dipeptidyl peptidase-IV-inhibitory activities

The antioxidant and antihypertensive activities of feather hydrolysates obtained with the bacterium Chryseobacterium sp. kr6 were investigated. Keratin hydrolysates were produced with different concentrations of thermally denatured feathers (10-75 g l(-1)) and initial pH values (6.0-9.0). Soluble proteins accumulated in high amounts in media with 50 and 75 g l(-1) of feathers, reaching values of 18.5 and 22 mg ml(-1), respectively, after 48 hours of cultivation. In media with 50 g l(-1) of feathers, initial pH had minimal effect after 48 hours. Maximal protease production was observed after 24 hours of cultivation, and feather concentration and initial pH values showed no significant effect on enzyme yields at this time. Feather hydrolysates displayed in vitro antioxidant properties, and optimal antioxidant activities were observed in cultures with 50 g l(-1) feathers, at initial pH 8.0, after 48 hours growth at 30°C. Also, feather hydrolysates were demonstrated to inhibit the angiotesin I-converting enzyme by 65% and dipeptidyl peptidase-IV by 44%. The bioconversion of an abundant agroindustrial waste such as chicken feathers can be utilized as a strategy to obtain hydrolysates with antioxidant and antihypertensive activities. Feather hydrolysates might be employed as supplements in animal feed, and also as a potential source of bioactive molecules for feed, food and drug development.

English

The protease producing bacterial strain Bacillus subtilis was isolated from sea water and identified by 16S rRNA sequencing. The strain named as B. subtilis NS. Optimization of the strain revealed that the most suitable nitrogen source to enhance protease production was beef extract. Among various carbon sources tested, maximum production of protease was registered in medium with added glucose. The effect of metals ions indicated that maximum protease production was observed in medium supplemented with magnesium chloride (MgCl). Investigating the effect of sodium chloride (NaCl) concentration on protease production revealed that 7% yielded higher protease production. The most suitable pH and temperature for maximum protease production revealed that pH 9 and a temperature of 40°C gave optimal protease production.   Key words:  Alkaline protease, Bacillus subtilis, media optimization, marine bacteria.

Cow Dung Substrate for the Potential Production of Alkaline Proteases by Pseudomonas putida Strain AT in Solid-State Fermentation

Cow dung and agroresidues were used as the substrates for the production of alkaline proteases by Pseudomonas putida strain AT in solid-state fermentation. Among the various substrates evaluated, cow dung supported maximum (1351±217 U/g) protease production. The optimum conditions for the production of alkaline proteases were a fermentation period of 48 h, 120% (v/w) moisture, pH 9, and the addition of 6% (v/w) inoculum, 1.5% (w/w) trehalose, and 2.0% (w/w) yeast extract to the cow dung substrate. The enzyme was active over a range of temperatures (50–70°C) and pHs (8–10), with maximum activity at 60°C and pH 9. These enzymes showed stability towards surfactants, detergents, and solvent and digested various natural proteins.

Optimization of Protease Production by Bacillus mojavensis A21 on Chickpea and Faba Bean

Response surface methodology (RSM) was employed to optimize the medium composition and culture conditions for the production of alkaline proteases by Bacillus mojavensis A21 on uncommon substrates: chickpea (CF) and faba bean (FF) flours. A significant positive influence of temperature, CF, FF, incubation time and inoculums size on the protease production was evaluated by Plackett Burman Design. Among these, CF was the most influential factor. The enhancement of protease to 9127 U/ml was achieved with the optimization procedure on the medium composed of (g/l): CF, 40; FF 30, NaCl 2.0; KH2PO4 1; K2HPO4 1; CaCl2, 0.1; MgSO4 0.1. The cultures were conducted for 72 hours with an IS of 2%, at 30&degC, an agitation speed of 150 rpm and an initial pH of 8.0. More interestingly, the optimization was accomplished using two cheap and local fermentation substrates, CF and FF, which could result in a significant reduction in the cost of medium constituents. The maximum alkaline protease production was 9127 U/ml after 72 h of incubation and showed 5-fold increase in protease production over the initial level.

OPTIMIZATION OF ALKALINE PROTEASE PRODUCTION BY <i>STREPTOMYCES AMBOFACIENS</i> IN FREE AND IMMOBILIZED FORM

Optimization of alkaline protease production by Streptomyces ambofaciens NRRL 2420 in free and immobilized form was investigated using submerged fermentation technique. The optimum conditions for maximum alkaline protease production 342 unit mL -1 were 30°C at pH 8.5 and incubation time 96 h in fr ee cell cultures using starch 20 g L -1 as carbon source and yeast extract 5 g L -1 as nitrogen source. The incubation time for the best yield of 344 unit mL -1 was reduced to 72 h under the optimized fermentati on conditions by immobilized cells adsorbed on synthetic cotton fibe rs. Data obtained during 5 reusable cycles showed h igher levels of enzyme in shorter time duration. Immobili zation of Streptomyces ambofaciens NRRL 2420 on synthetic cotton fiber permit repeated reuse of the cells under the optimized fermentation conditions.

Streptomyces flavogriseus HS1: isolation and characterization of extracellular proteases and their compatibility with laundry detergents.

The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5–11) and temperature (25–70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca2+ and Mg2+. EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level.

Production and biochemical characterization of protease from Aspergillus oryzae: An evaluation of the physical–chemical parameters using agroindustrial wastes as supports

Abstract This study reports the production optimization and the biochemical characterization of a partially purified protease obtained from Aspergillus oryzae LBA 01 under solid state fermentation (SSF) using different agroindustrial wastes as matrix supports. The wastes included wheat bran, soybean meal and cottonseed meal. All of them were characterized in terms of water absorption index, chemical composition and granulometric distribution. The substrates with highest water absorption index and more heterogeneous granulometric distribution have positively influenced on protease production. Some cultivation parameters were studied and the results showed that the optimum fermentation medium was composed of wheat bran, 2.0% (w/w) peptone and 2.0% (w/w) yeast extract, and the conditions for maximum protease production were an initial moisture content of 50.0%, an inoculum level of 10 7 spores g −1 and an incubation at 23 °C. The biochemical characterization using experimental design showed that the enzyme was most active over the pH range 5.0–5.5 and was stable from pH 4.5 to 6.0, indicative of an acid protease. The optimum temperature range for activity was 55–60 °C, and the enzyme was stable at 35–45 °C. The results showed that wheat bran have great potential as support matrix for protease production by A. oryzae LBA 01 in SSF.

Production, partial purification and characterization of protease from a phytopathogenic fungi Alternaria solani (Ell. and Mart.) Sorauer

An alkaline serine protease producing strain Alternaria solani was optimized for its enzyme production under submerged conditions. The maximum production of protease by A. solani was achieved by using sodium nitrate at the optimum concentration of 0.2% w/v. A. solani produced higher quantities (3.75 [unit/mg of protein]) of an inducible extracellular proteases on day 9 after incubation in czapek's dox broth medium amended with 1% casein as an inducer at pH 8.5, temperature 27 °C and 3% sucrose as carbon source. Extracellular proteases were precipitated by ammonium sulphate saturation (80%) method and purified on Sephadex G-100 column chromatography. The molecular mass of SDS-PAGE and Sephadex G-100 Column Gel permeation chromatography purified protease was estimated to 42 kDa. In addition, trypsin digestion of 42 kDa protein band was carried out and analyzed by MALDI-TOF for the identification of protease. The sequence IKELATNGVVTNVK (378-391) segment of the alkaline serine protease was found by using MS/MS spectrum at 1485 m/z from the purified fraction. It showed optimal activity at 50 °C and pH 9-10 and broad pH stability between pH 6-12. The protease activity was inhibited by phenyl methyl sulfonyl fluoride (PMSF), all the results indicated that the presence of a serine residue in the active site and is thus most likely a member of the serine protease family. This may function as a virulence protein during pathogenesis by A. solani. The results suggested that the presence of appreciable extracellular proteolytic activity in filamentous fungi may serve as a marker of their phytopathogenicity.

Protease Production by <i>F</i><i>usarium </i><i>o</i><i>xysporum</i> in Solid- State Fermentation Using Rice Bran

Agroindustrial wastes constitute valuable source for microbial cultivation and enzyme production. An attempt was made to study protease production by Fusarium oxysporum on agroindustrial waste rice bran under solid-state fermentation. Rice bran obtained from rice mill has proved to be efficient substrate for enzyme production by F. oxysporum. To study the maximum enzyme production the process parameters of fermentation where optimized. Maximum protease production i.e. (70.5U/g) was obtained with an initial moisture content of 50% (w/w) on incubation period of 72h at 35°C temperature with initial pH of substrate 7.0. The above data revealed that agroindustrial waste rice bran and optimized condition of solid-state fermentation can be successfully employed for protease production by Fusarium oxysporum.

Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments

Abstract Protease producing halotolerant bacterium was isolated from saltern pond sediment (Tuticorin) and identified as Bacillus licheniformis (TD4) by 16S rRNA gene sequencing. Protease production was enhanced by optimizing the culture conditions. The nutritional factors such as carbon and nitrogen sources, NaCl and also physical parameters like incubation time, pH, agitation, inoculum size were optimized for the maximum yield of protease. Studies on the effect of different carbon and nitrogen sources revealed that xylose and urea enhances the enzyme production. Thus, with selected C–N sources along with 1 M NaCl the maximum protease production (141.46 U/mg) was obtained in the period of 24 h incubation at pH 8 under 250 rpm compared to the initial enzyme production (89.87 U/mg).

STATISTICAL MODELING AND OPTIMIZATION OF ALKALINE PROTEASE PRODUCTION FROM A NEWLY ISOLATED ALKALOPHILIC Bacillus SPECIES BGS USING RESPONSE SURFACE METHODOLOGY AND GENETIC ALGORITHM

A new hyperactive alkalophilic bacterial strain (Bacillus sp. BGS) was isolated from samples collected from soil that received the effluent of a milk processing industry located in Madurai, Tamilnadu, India, and this bacterial strain was used for the production of alkaline protease. Four out of eight variables, such as molasses, peptone, pH, and inoculum size, have been identified through Plackett–Burman (PB) design and used for the alkaline protease production. These significant variables were further optimized through a hybrid system of response surface methodology (RSM) followed by genetic algorithm (GA). The optimal combination of media components and culture conditions for maximal protease production was found to be 16.827 g/L of peptone, 1.128% (v/v) of molasses, pH value of 11, and 2% (v/v) of inoculum size. A 6.36-fold increase in protease production was achieved through the RSM–GA hybrid system. The protease activity increased significantly with an optimized medium (2,992.75 U/mL) as opposed to an unoptimized basal medium (470.35 U/mL).

Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8–10, expressed their maximum activities at pH10 and temperature range of 30–50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51–97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

Isolation and Characterization of a Feather Degrading Alkalophilic Streptomyces sp. TBG-S13A5 and its Keratinolytic Properties

Keratinases are of particular interest because of their action on insoluble keratins and generally on a broad range of protein substrates. Alkalophilic and neutrophilic actinomycete strains isolated from different soil samples, rich in keratinaceous substances were screened for keratinolytic activity. An alkalophilic isolate, TBG-S13A5, was found to possess good keratinolytic activity and was able to utilize feather as the sole carbon and nitrogen source. TBG-S13A5 exhibited an off-white aerial mass color with a rectus-flexibilis type of spore chain. The morphological, microscopical and biochemical characters were comparable with that of Streptomyces albidoflavus. Fatty acid methyl ester profiling (FAME) and 16S rDNA sequence analysis confirmed its identity as a strain of S. albidoflavus. Under submerged fermentation conditions, maximum protease production was recorded on the 5th day of incubation at 30℃, using basal broth of pH 9.0 with 0.25% (w/v) white chicken feather. This strain could affect feather degradation when the initial pH was 8 and above and maximum protease production was recorded when the initial pH was around 10.5. The effectiveness of the crude enzyme in destaining and leather dehairing were also demonstrated.

Optimization of cultural conditions for the production of an extracellular protease by Pseudomonas species

The aim of the study was to identify new sources of protease and a protease producing bacteria was isolated from slaughter house soil and identified as Pseudomonas species. The protease production efficiency of the organism was measured with different environmental and nutritional parameters. Optimization of the fermentation medium for maximum protease production was carried out. The culture condition like temperature, pH, incubation time, carbon and nitrogen sources were optimized. The optimum condition found for protease production was 30°C at pH 7 in the medium for 48 hour. Carbon source casein and nitrogen source beef extract stimulated the production of protease. Protease production was higher with immobilized cells. Pseudomonas species can be used profitably for the large scale production of protease to meet the present day demand of the industrial sector.DOI: http://dx.doi.org/10.3329/icpj.v2i1.12870 International Current Pharmaceutical Journal 2012, 2(1): 1-6

Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3

Applications of keratinases demand efficient production and downstream processing. This work reports the production, purification and partial characterization of a keratinolytic protease from Serratia marcescens P3. The influence of temperature, initial pH and feather meal concentration on enzyme production was investigated using response surface methodology. Maximal protease production (12.5UmL−1) was observed at 18–30°C, 10–24gL−1 feather meal, and pH values of 6.5–8.5. The enzyme was recovered from culture supernatants using aqueous two-phase systems (ATPSs), and higher yields (68%) were obtained in polyethylene glycol (PEG) 4000-sodium citrate and PEG 4000-ammonium sulfate systems. These ATPS were effective for enzyme purification, resulting in an electrophoretically homogeneous protein of 53kDa. Sequencing of tryptic peptides identified this enzyme as a metalloprotease belonging to the serralysin family of metzincins. The purified enzyme showed optimal activity at 40–45°C and pH 6.5, being also active toward azokeratin.

Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor

We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0-8.0 and 8.0-10, respectively. Maximum production of proteases was observed at an incubation temperature of 37°C while that of alpha amylase was observed at 40°C. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.

Cow dung as a novel, inexpensive substrate for the production of a halo-tolerant alkaline protease by Halomonas sp. PV1 for eco-friendly applications

The production of a halo-tolerant-alkaline protease by Halomonas sp. PV1 under solid state fermentation was optimized. Among the substrates evaluated, cow dung supported the maximum protease production (1351U/g) when compared with wheat bran (1013U/g). Process parameters such as, the fermentation period (72h), pH (8.0), initial moisture (140%, v/w) and the inoculum level (15%, v/w) were optimized. The optimum enzyme production was achieved with 1.5% (w/w) xylose and a 1.25% (w/w) yeast extract. The partially purified protease enzyme was active over a temperature range of 30–50°C and a pH range of 7–10. The protease was remarkably stable on sodium dodecyl sulfate and on various commercial detergents. The enzyme effectively dehaired goat hides, and makes it a potential source of alkaline protease. This substrate may have wide spread applications in enzyme bioprocesses like wheat bran.

Isolation of alkaline protease from Bacillus subtilis AKRS3

This research study was mainly focused on phenotypic, biochemical characterization, 16s rRNA sequence based species level identification of isolate and determination of the higher production of alkaline protease through optimization study (carbon, nitrogen, incubation period, temperature, pH and sodium chloride concentration), production by submerged fermentation and analytical studies (protease assay, protein and biomass estimation). The produced crude enzyme was been used for dehairing activity on goat and sheep hides. Primary screening was achieved by skim milk casein hydrolysis method. Microbiological, biochemical characterization and 16S rRNA phylogenetic analysis revealed that isolated bacterium was Bacillus subtilis AKRS3 with an optimum alkaline protease producing temperature, 37°C and pH 9.0. The maximum alkaline protease production was achieved at 24 h of incubation period. Among various nitrogen (organic and inorganic) sources, beef extract was found to be the best inducer for alkaline protease in the concentration of 1.5% as was reported for the maximum alkaline protease production. Effect of carbon sources for example xylose, on protease production proved high protease production than the other tested carbon sources and subsequently 2% concentration registered an optimum to enhance the protease production. The halotolerancy of B. subtilis AKRS3 for alkaline protease production indicated that 3% of sodium chloride was optimum to yield maximum protease activity. During production, agitation rate was 250 rpm at air flow rate of 1 VVM. Maximum protease activity of 42.7556 U/ml was observed at the end of 24 h cell free supernatant of fermentation broth. Crude alkaline protease was most active at 55°C, pH 9 with casein as substrate. According to our knowledge, this study demonstrated the first report on alkaline protease producing B. subtilis AKRS3 isolated from fish waste. The produced enzyme could be effectively used to remove hair from goat and sheep hide indicating its potential application in leather processing industry. Key words : Bacillus subtilis AKRS3, 16S rRNA sequence, alkaline protease, submerged fermentation (SmF), dehairing activity.

Isolation and Molecular Characterisation of Alkaline Protease Producing Bacillus thuringiensis

Proteases are of particular interest because of their action on insoluble keratin substrates and generally on a broad range of protein substrates. Proteases are one of the most important groups of industrial enzymes used in detergent, protein, brewing, meat, photographic, leather, dairy, pharmaceutical and food industry. In the present study, the organism isolated from the protein rich soil sample was identified by biochemical and molecular characterisation as Bacillus thuringiensis and further optimum conditions for alkaline protease synthesis were determined. The growth conditions for B. thuringiensis was optimised by inoculating into yeast extract casein medium at different pH and incubating at different temperatures. The maximum protease production occurred at pH 8 and at 37 °C. B. thuringiensis showed proteolytic activity at various culture conditions. Optimum conditions for the protease activity were found to be 47 °C and pH 8. In the later stage, the blood removing action of crude and partially purified protease was found to be effective within 25 min in the presence of commercial detergents indicating the possible use of this enzyme in detergent industry. Enzyme also showed good activity against hair substrate keratin and can be used for dehairing.

Optimization of Culture Conditions for Production of Protease by Two Bacterial Strains, Bacillus licheniformis BF2 and Bacillus subtilis BH4 Isolated from the Digestive Tract of Bata, Labeo bata (Hamilton)

The culture conditions for extracellular protease production by two bacterial strains, Bacillus licheniformis BF2 and Bacillus subtilis BH4 isolated from the foregut and hindgut regions, respectively, of bata (Labeo bata, Cyprinidae) were optimized to obtain maximum level of protease. Both strains were cultured in TSA broth for 24 h at 37 ± 2 °C. This was used as the inoculum for the production medium. The fermentation medium was seeded with 1.0, 2.0, 3.0, 4.0, and 5.0 % inoculum (TSA) and incubated in static culture at 37 °C to standardize the inoculum size for fermentation. The effects of fermentation condition, moisture, pH, temperature, inoculum size, and nitrogen sources on protease production by the isolated bacterial strain were studied. The strain, BH4, produced more protease in comparison to the strain BF2 isolated from the same fish in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 6.0–6.5. Maximum protease production by both the strains was obtained at 40 °C. To standardize the fermentation period for protease production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points. Beef extract as the nitrogen source was most effective in optimizing enzyme production by both the isolated strains. The information generated from the present investigation will help to standardize the requirements for optimum production of protease by protease-producing fish gut bacteria.