Production Of Extracellular Alkaline Protease Research Articles

Efficient production of extracellular alkaline protease in Bacillus amyloliquefaciens by host strain construction

Bacillus amyloliquefaciens is an important industrial microorganism, which can be applied as a protein expression host. However, the extracellular protein expression ability of B. amyloliquefaciens is still low. Hence, it is necessary to develop a B. amyloliquefaciens host with a high extracellular protein expression capacity. In this study, B. amyloliquefaciens HZ-12 was the original strain, then seven important extracellular protease genes (epr, nprE, aprE-a, mpr, pbpF, vpr, ykct1), one important intracellular protease gene (aprX) and two redundant proteins genes (htrB, hag) were deleted in HZ-12, resulting in B. amyloliquefaciens BAX-10. In order to evaluate the extracellular protein expression ability of BAX-10, the alkaline protease gene aprE from Bacillus subtilis D7 was expressed in BAX-10. After fermentation, the alkaline protease activity of BAX-10/aprE reached 666.82 U/mL, which was 57% higher than that of the control strain HZ/aprE. Moreover, the deletion of the ten genes had no negative effect on the growth of B. amyloliquefaciens. In summary, B. amyloliquefaciens BAX-10 could be used as a potential platform host for expression of target extracellular proteins.

Effect of alkaline protease produced from fish waste as substrate by Bacillus clausii on destaining of blood stained fabric

Alkaline protease or peptidases are the largest groups of enzymes in the biological industry with a variety of application in manufacturing units used in the process of substrate stabilization, dehairing, diagnosis, extraction, food production, destaining, etc., where the pH of the environmental conditions remain above neutral pH. Because of these wider applications of alkaline proteases in industries, their demand is increasing to compete with their chemical counterpart. An alkaline tolerant bacterial strain Bacillus clausii wasisolated from fish waste and used for mass production of alkaline protease using fish waste homogenate as media. Preliminary study on optimization of conditions for the mass production carried out. The optimum temperature for production ranges between 25°C and 35°C and pH determined as 9. The mass production of extracellular alkaline protease carried out using mobilized and immobilized cells of B. clausii at optimized condition using production media, the mixture of production media and fish waste homogenate and in nutrient broth. The recorded results showed that the maximum enzyme production obtained with immobilized cells in nutrient broth media and followed by fish waste homogenate media of 8900 U/ml and 8600 U/ml. Purification protease enzyme yielded 0.35 g/ml from the production media . Bloodstained cloth treated with immobilized enzyme removed the stain completely compared to treatment with non-immobilized enzyme and commercially used detergent. So, the current study suggests the usage of microbial alkaline protease in household detergents to replace the usage of synthetic detergents and save the environment from chemical pollutants.

Optimized production of extracellular alkaline protease from Aspergillus tamarii with natural by-products in a batch stirred tank bioreactor

Proteolytic enzymes are one of the significant commercially manufactured enzymes. The manufacture of extracellular alkaline protease by Aspergillus tamarii MTCC5152 was explored using several agricultural by-products as substrates viz., cottonseed meal, wheat bran, skimmed milk and soya flour in submerged fermentation, were found to be efficient for enzyme production and commercially significant. Response surface methodology (RSM) is a statistics-based experimental design, sourced to explore the impact of physical parameters on the manufacture of protease from A. tamarii in a batch stirred tank bioreactor (STBR). The four substantial variables (pH, temperature, inoculum size, and agitation) were carefully chosen for optimization analyses and the statistical pattern was created using a central composite design and the quadratic model has been developed. The optimum conditions for protease production (1.51 U mL−1) where: pH 6.4, temperature 27 °C, inoculum size 2.6%, and agitation 327 rpm. The analysis revealed that the anticipated values were in accord with trial data with a correlation coefficient of 0.969.

English

The production of extracellular alkaline protease by Bacillus subtilis was studied with submerged fermentation. A new strain of Bacillus sp. was isolated from alkaline soil, which was able to produce extracellular alkaline protease. The production of alkaline protease involved the use of agricultural or animal wastes at pH 8 and temperatures at 37°C. Results showed that growing B. subtilis sub sp. subtilis under optimized growth resulted in production of alkaline protease with enzyme activity of 1412.5 U/ml, while with pomegranate peel at a concentration of 3%, the enzyme activity reached 3600 U/ml; further increase in pomegranate concentration did not however, lead to additional enzyme activity. Among various nitrogen sources, yeast extract was found to be the best inducer of alkaline protease. Among metal salts, KNO3 and NH4Cl were found to increase protease production. The maximum enzyme production (3600 U/ml) was observed with pomegranate peels of fermentation medium in the presence of yeast extract, potassium nitrate and ammonium chloride. Key words: Production, alkaline protease, Bacillus subtilis, animal wastes, enzyme activity.

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

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

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

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. 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). 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 of extracellular alkaline protease from Bacillus subtilis RSKK96 with solid state fermentation

The production of extracellular alkaline protease by producing Bacillus subtilis RSKK96 was studied with solid state fermentation (SSF). Different agro residues as substrate were studied for enzyme production. The highest enzyme production was expressed with lentil husk as units per mass of dry substrate (3937.0 U/mg). Production parameters were optimized as incubation time 120 h, extraction medium Triton-X100 1%, initial moisture content 30%, initial pH 9.0. The high level of alkaline protease was obtained in the medium containing arabinose followed by lactose, galactose, and fructose. Among various nitrogen sources, beef extract was found to be the best inducer of alkaline protease, while other nitrogen sources repressed enzyme production. Among metal salts FeSO4.7H2O and MgSO4.7H2O was found to increase protease production. The maximum enzyme production (5759.2 U/mg) was observed with lentil husk in 1000 mL of fermentation medium volume.

Optimizing some factors affecting alkaline protease production by a marine bacterium Teredinobacter turnirae under solid substrate fermentation

Production of extracellular alkaline protease by a shipworm bacterium Teredinocabcter turnirae, under solid substrate fermentation (SSF) was optimised. Soybean was used as a sole carbon and/or nitrogen source. The maximum protease yield (1950 U/ml) was achieved with optimised parameters such as coarse size of soybean (2 mm), concentration of soybean 1% (w/v), unadjusted pH of the medium 7.34, inoculum level 2.5% (v/v) and agitation rate 120 rpm. Sixty percent of higher production was achieved after optimising the process parameters.

Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14

Statistical optimization of culture conditions for production of a widely suited detergent protease from Bacillus sp. RGR-14 was carried out using a two-step approach. A quick identification of the important factors with simple screening experiment was followed by application of complex response surface design for further optimization. The production of extracellular alkaline protease by Bacillus sp. was favored in the presence of complex carbon and nitrogen sources, viz. starch, casamino acid and soybean meal. A reduced quadratic model was found to fit the alkaline protease production. Response surface analysis revealed the significant role of phosphate ions in determining alkaline protease production. A steep, stretched out response surface showed direct relation between the level of protease production and casamino acid and starch concentration in the medium. A 12.85 fold increase in protease production could be obtained within the design space. Protease production was found to be repressed in the presence of high concentrations of casamino acid. The model could be validated in up to 2 l shake flasks (3914 U ml −1). The same statistical design could explain economic protease production in cost-effective medium as well.

Production of extracellular alkaline protease by immobilization of the marine bacterium Teredinobacter turnirae

Cells from a marine bacterium, Teredinobacter turnirae cells were immobilized in calcium alginate beads and used for alkaline protease production. The maximum protease activity was obtained at 3% (w/v) sodium alginate and 3% CaCl 2 concentrations with a 1/2 cell/alginate ratio, i.e. 2400 U/ml. There was no significant difference in the maximum protease activity between three bead sizes used. A drastic fall in protease production was observed when the beads were treated with glutaraldehyde. The beads were used for eight successive fermentation batches each lasting 72 h. It was also observed that there was a ∼3.5-fold increase in volumetric productivity of protease after the fourth cycle.

Optimizing some factors affecting protease production under solid state fermentation

Production of extracellular alkaline protease by a locally isolated fungal species, Rhizopus oryzae, under solid state fermentation was optimized. The maximum enzyme activity under the optimum conditions of temperature (32 °C), relative humidity (90%–95%), spore count (∼2 × 105/g wheat bran), moisture content of solid substrate (140%) adjusted suitably with salt solution (M-9) of pH 5.5 was 341 unit/g wheat bran.

Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT

The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector. When B. subtilis MT-2 (Npr- [deficiency of neutral protease] Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. In contrast, when B. subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity. After construction of the deletion plasmids, DNA sequencing was done. A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244). A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame. A B. subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B. subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division. When B. stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division. These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B. subtilis. However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products. Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366). We discuss the possibility that the membrane protein DegT functions as a sensor protein and transfers the signal of environmental stimuli to the regulatory region of target genes to activate or repress transcription of the genes.