Extracellular Alkaline Protease Research Articles

Production and characterization of an organic solvent activated protease from haloalkaliphilic bacterium Halobiforma sp. strain BNMIITR

An unusual haloalkaliphilic bacterium known as Halobiforma sp. strain BNMIITR, which was noticed to produce an extracellular alkaline protease, was found in a soil sample from Northern India's Sambhar Lake. On the generation of protease, the effects of dietary elements including nitrogen and carbon sources, amino acids, and growth conditions like temperature and pH were investigated. When low-cost agricultural by-products were employed as nitrogen sources, the manufacturing of enzymes was significantly boosted. In the present study, protease production was enhanced by 2.94 fold and 2.17 fold. By solvent precipitation and Hydrophobic interaction chromatography (HIC) on Phenyl Sepharose 6 Fast Flow matrix, the enzyme was purified 31.67 fold. It was determined that the apparent molecular mass was 21 kDa. The pH range where the enzyme was most stable was 6.0–12.0, with a temperature of 50 °C as optimum. When there was alkaline earth metals and heavy metals, protease was discovered to be active. It was evident that the enzyme was a serine type of protease because it was active in the presence of a variety of surfactants, oxidizing and reducing chemicals, and phenylmethylsulfonyl fluoride (PMSF) completely inhibited activity. Enzyme exhibited a wide range of substrate specificity. Amazingly, enzyme remained stable both in polar and nonpolar solvents. The most interesting aspect of this enzyme is enhanced activity in polar solvents like dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). It was discovered that the protease was stable and compatible with a number of widely available detergents.

Extracellular activity of proteases from Yarrowia lipolytica IPS21 as a function of the carbon and nitrogen source

Abstract The yeast strain Yarrowia lipolytica IPS 21 was tested for its ability to produce the protease enzyme on analytically pure carbon sources as well as on waste carbon sources. It was confirmed that the yeast Y. lipolytica IPS21 can have a higher proteolytic activity in the presence of waste carbon sources in chrome-tanned leather shavings (CTLS) than on yeast extract alone. This is confirmed by the high concentration of amino acids in samples with CTLS, suggesting increased degradation of CTLS by Y. lipolytica or secretion of proteases into the medium. It was also confirmed that metals accumulate mainly in the biomass and not in the supernatant. The biomass was also found to contain high levels of Ca, K and P, which are essential for plant growth. These results show that Y. lipolytica strain IPS21 can be used for the production of extracellular alkaline proteases and for the degradation of protein waste.

A systematic analysis of the subtilase gene family and expression and subcellular localization investigation of anther-specific members in maize

Subtilases (SBTs), also known as Subtilisin-like serine proteases, are extracellular alkaline protease proteins. SBTs function in all stages of plant growth, development and stress responses. Maize (Zea mays L.) is a crop widely used worldwide as food, feed, and industrial materials. However, information about the members and their functions of the SBT proteins in maize is lacking. In this study, we identified 58 ZmSBT genes from the maize genome and conducted a comprehensive investigation of ZmSBTs by phylogenetic, gene duplication event, gene structure, and protein conserved motif analyses. The ZmSBT proteins were phylogenetically classified into seven groups, and collinearity analysis indicated that many ZmSBTs originate from tandem or segmental duplications. Structural and homolog protein comparison revealed ZmSBTs have conserved protein structures with reported subtilase proteins, suggesting the conserved functions. Further analysis showed that ZmSBTs are expressed in different tissues, and many are responses to specific abiotic stress. Analysis of the anther-specific ZmSBT genes showed their expression peaked at different developmental stages of maize anthers. Subcellular localization analysis of selected maize ZmSBTs showed they are located in different cellular compartments. The information provided in this study is valuable for further functional study of ZmSBTs.

Identification, Isolation, Cloning, and Expression of a New Alkaline Serine Protease Gene from Native Iranian Bacillus sp. RAM 53 for Use in the Industry.

Proteases are one of the most important and widely applicable proteolytic enzymes that are used in various industries. The aim of this study was to identify, isolate, characterize, and clone the new extracellular alkaline protease from the native bacterium Bacillus sp. RAM53 that was isolated from rice fields in Iran. In this study, first, the primary assay of protease production was performed. The bacteria were cultured in a nutrient broth culture medium at 37° C for 48h, and then, the enzyme extraction was performed. Enzyme activity was measured by standard methods in the range of 20 to 60°C and the range of pH 6.0 to 12. Degenerate primers were designed to alkaline protease gene sequences. The isolated gene was cloned into the pET28a+ vector, the positive clones were transferred to Escherichia coli BL21, and the expression of the recombinant enzyme was optimized. The results showed that the optimum temperature and pH of the alkaline protease were 40° C and 9.0, respectively, and were stable at 60° C for 3h. The molecular weight of the recombinant enzyme was 40kDa in SDS-PAGE. The recombinant alkaline protease was inhibited by the PMSF inhibitor, indicating that the enzyme was serine protease. The results showed that the sequence alignment of the enzyme gene with the other alkaline protease gene sequences related to Bacillus was 94% identity. The result of Blastx showed about 86% identity to the S8 peptidase family in Bacillus cereus and Bacillus thuringiensis and other Bacillus species. The enzyme may be useful for various industries.

Optimization, partial purification, and characterization of a novel high molecular weight alkaline protease produced by Halobacillus sp. HAL1 using fish wastes as a substrate

BackgroundHydrolytic enzymes from halophilic microorganisms have a wide range of industrial applications. Herein, we report the isolation of Halobacillus sp. HAL1, a moderately halophilic bacterium that produces a novel high molecular weight extracellular alkaline protease when grown in fish processing wastes as a substrate. ResultsResults showed that the isolated strain belonged to the genus Halobacillus, and it was designated as Halobacillus sp. HAL1 with the GenBank accession number OK001470. The strain secreted an extracellular alkaline protease, and the highest yield was obtained when it was grown in a medium with fish wastes substrate as the sole nutritional source (10 g/L) and incubated at 25 °C under shaking conditions. The enzyme was partially purified by Sephadex G-100 column chromatography. Zymographic analysis showed two casein degrading bands of about 190 and 250 KDa. The optimum enzyme activity was at a temperature of 50 °C at pH 8. The proteolytic activity was enhanced in the presence of metal ions (Ca2+, Mg2+, and Mn2+), surfactants (Tween 80, SDS, and Triton-X100), H2O2, and EDTA. ConclusionOur study indicates that Haobacillus sp. HAL1 is a moderately halophilic strain and secrets a novel high molecular wight alkaline protease that is suitable for detergent formulation.

Comparative Analysis of the Alkaline Proteolytic Enzymes of Yarrowia Clade Species and Their Putative Applications

Proteolytic enzymes are commercially valuable and have multiple applications in various industrial sectors. The most studied proteolytic enzymes produced by Yarrowia lipolytica, extracellular alkaline protease (Aep) and extracellular acid protease (Axp), were shown to be good candidates for different biotechnological applications. In this study, we performed a comprehensive analysis of the alkaline proteolytic enzymes of Yarrowia clade species, including phylogenetic studies, synteny analysis, and protease production and application. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we reconstructed the evolutionary scenario of the XPR2 gene for species of the Yarrowia clade. Furthermore, except for the proteolytic activity of the analyzed Yarrowia clade strains, the brewers' spent grain (BSG) was used as a substrate to obtain protein hydrolysates with antioxidant activity. For each culture, the degree of hydrolysis was calculated. The most efficient protein hydrolysis was observed in the cultures of Y. lipolytica, Y. galli, and Y. alimentaria. In contrast, the best results obtained using the 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) method were observed for the culture medium after the growth of Y. divulgata, Y. galli, and Y. lipolytica on BSG.

Production and characterization of crude proteases from Halobacillus salinus strain DZ28 newly isolated from salt lake sediments in Algeria and their use as detergent bioadditives

New Algerian Gram-positive, rod-shaped, endospore-forming, salt-philic bacteria (DZ28 strain) that overproduce extracellular alkaline proteases have been isolated from salt lake deposits in Lake Oubeira, El Taref. Strain DZ28 was assigned as Halobacillus salinus DZ28 on the basis of phenotypic properties and 16S rDNA gene sequencing (ripotyping). The maximum protease activity registered after 36 hours of incubation in optimized medium at 30 ° C was 19,000 U / ml in a shaking bottle culture at 160 rpm. The crude extract protease showed optimal activity at 60 ° C temperature and pH 12. It is actively inhibited by PMSF and DIFP, indicating that it belongs to the serine protease family. Interestingly, the crude extract protease was not only very stable to nonionic surfactants and oxidants, but also showed high stability and compatibility with some commercial detergents. It retaining more than 100% of its initial activity after pre-incubation for 1 h at 40°C with ISIS, followed by Pril (98%), Tide (95%) and Dixan (90%). More curiously, the wash overall performance evaluation discovered that it may dispose of blood-stains remove at 40°C for 1 h with low supplementation (500 U/mL). This is the first report of a protease from Halobacillus salinus and has potential as a promising candidate for future applications as a bioadditive for detergent formulations.

A comprehensive proteomics analysis of the response of Pseudomonas aeruginosa to nanoceria cytotoxicity

The increased commercial use and spread of nanoceria raises concerns about the risks associated with its effects on living organisms. Although Pseudomonas aeruginosa may be ubiquitous in nature, it is largely found in locations closely linked with human activity. P. aeruginosa san ai was used as a model organism for a deeper understanding of the interaction between biomolecules of the bacteria with this intriguing nanomaterial. A comprehensive proteomics approach along with analysis of altered respiration and production of targeted/specific secondary metabolites was conducted to study the response of P. aeruginosa san ai to nanoceria. Quantitative proteomics found that proteins associated with redox homeostasis, biosynthesis of amino acids, and lipid catabolism were upregulated. Proteins from outer cellular structures were downregulated, including transporters responsible for peptides, sugars, amino acids and polyamines, and the crucial TolB protein of the Tol-Pal system, required for the structural formation of the outer membrane layer. In accordance with the altered redox homeostasis proteins, an increased amount of pyocyanin, a key redox shuttle, and the upregulation of the siderophore, pyoverdine, responsible for iron homeostasis, were found. Production of extracellular molecules, e.g. pyocyanin, pyoverdine, exopolysaccharides, lipase, and alkaline protease, was significantly increased in P. aeruginosa san ai exposed to nanoceria. Overall, nanoceria at sublethal concentrations induces profound metabolic changes in P. aeruginosa san ai and provokes increased secretion of extracellular virulence factors, revealing the powerful influence this nanomaterial has on the vital functions of the microorganism.

PHYSICAL AND CHEMICAL CHARACTERIZATION OF ALKALINE PROTEASE FROM BACILLUS SUBTILIS VBC7 USING AGRO WASTE AS SUBSTRATE

Alkaline proteases are the most important group of industrial enzymes that hydrolyse the peptide bond of proteins into small peptides. The industrial demand of alkaline protease predominantly from the microbial origin has been recently increased and enhances the research for alkaline protease with high stability at extreme industrial conditions. Thus, this study is aimed to characterize the alkaline protease from bacterial isolate, Bacillus subtilis VBC7 screened from dairy waste dumped soil. Extracellular alkaline protease production was carried out in alkaline broth by submerged fermentation. The production medium with 10% ground nut extract at pH 10 and 40 °C enhanced the alkaline protease activity (712 U/mL) than other wastes such as coconut pulp extract and sesame seed extract. This optimized media increased the bacterial growth rate and activity of alkaline protease compared to the unsupplemented basal medium. Further, alkaline protease was partially purified and assessed their molecular weight (~30 k Da) in 12 % SDS -PAGE. The enzyme activity was observed at pH 10 and 40 °C and stable over a wide range of pH (5-12), and temperatures (10-70 °C). In addition, enzyme activity was stimulated in the presence of Mg2+, Ca2+ and Mn2+ and was unpretentious after treating with surfactants (SDS, Triton X-100, Tween 80), organic solvents (ethanol, methanol, chloroform, acetone and hexane) and protease inhibitors (EDTA and β-mercaptoethanol). These compatible features could lead the enzyme as a potential candidate among the industrial sectors.

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.

Screening and optimization of candidate alkaline protease for dehairing potential from marine Bacillus paramycoides M2

Alkaline proteases are active from neutral to alkaline pH range and have extensive applications in detergent and leather industries. In the present research, bacteria isolated from marine water samples were screened for proteolytic activity. Among the isolates, M2 showed maximum proteolysis with a clear zone when cultured on skim milk agar plates at 37°C for 24 h. Molecular identification using 16S rRNA sequencing and phylogenetic analysis revealed that M2 has sequence identity (99.93%) to Bacillus paramycoides. SEM analysis was carried for determining the morphology of M2 and also for enzyme treated skin. FAME analysis using GCMS was performed for the determination of fatty acids in the strain. The selected isolate was inoculated into protease production medium under submerged fermentation conditions at 37ºC for 48 h with a constant agitation of 120 rpm. Protease activity was determined under varying conditions of pH, incubation temperature, carbon and nitrogen sources, metal ions and NaCl (1- 5%) using casein as substrate. The isolate M2 utilized molasses and peptone as carbon and nitrogen sources for better alkaline protease production at 40°C and pH 10 under optimal conditions. The dehairing experiments with M2 alkaline protease revealed dehairing efficacy of protease over chemical treatment. Hence, extracellular alkaline protease from M2 isolate could find potential application in leather processing industries and can be exploited commercially.

Characterization of the genome and serine protease of a novel Bacillus subtilis isolate.

Current study was undertaken to carry out the genome-wide analysis of a multipotent isolate from desert soil which was previously identified as Bacillus tequilensis based on 16S rDNA analysis. This study also aims to characterize the serine protease and its biocatalytic potentials implying a combination of empirical and in-silico approaches. Next generation sequencing and short read de novo assembly generated the 4,235,084bp draft genome of Bacillus sp. ZMS-2. Genome sequence analysis by digital DNA:DNA hybridization (dDDH) and average nucleotide identity classified the isolate as Bacillus subtilis ZMS-2 (Bioproject ID: PRJNA691551). Genome annotation revealed 10 antibiotic resistance genes, 8 antibiotic/antifungal gene clusters and 25 genes encoding proteases including subtilisin E, an extracellular alkaline protease. This extracellular protease (ZMS-2 protease) was produced using a statistically optimized medium, purified partially and characterized as alkaline serine protease. The partially purified ZMS-2 protease (780U/mL) showed a 21mm zone of casein hydrolysis and dehaired goat skin by pulling out hair with roots. These catalytic potentials of ZMS-2 protease were further confirmed using scanning electron microscopy of casein beads and dehaired skin. The study concludes B. subtilis ZMS-2 as a potent producer of a protease with promising potentials of commercial importance.

Adsorption of extracellular proteases and pyocyanin produced by Pseudomonas aeruginosa using a macroporous magnesium oxide-templated carbon decreases cytotoxicity

Pseudomonas aeruginosa is one of the most common pathogens isolated in clinical settings and produces a wide range of extracellular molecules that contributes to the virulence. Chemotherapy options to prevent and treat P. aeruginosa infections are limited because this pathogen is highly and innately resistant to some classes of conventional drugs. Alternative methods to conquer P. aeruginosa, including multidrug resistant strains, are being investigated. This study showed that a macroporous magnesium oxide (MgO)-templated carbon material (MgOC150) attenuates the toxicity of this bacterium in human epithelial cells. A proteomic analysis revealed that MgOC150 adsorbs some extracellular proteases, including elastase (LasB) and alkaline protease (AprA), required for the virulence of P. aeruginosa, which decreases the accumulation of these molecules. MgOC150 also adsorbed pyocyanin, which is another molecule involved in its pathogenesis, but is a nonprotein small-sized molecule. These results suggest a potency of MgOC150 that suppresses the virulence of P. aeruginosa. MgOC150 has been used for industrial purposes, as an electrode catalyst and a bioelectrode and for enzyme immobilization. Thus, MgOC150 could be beneficial for developing novel anti-Pseudomonas therapy.

Multipotential Alkaline Protease From a Novel Pyxidicoccus sp. 252: Ecofriendly Replacement to Various Chemical Processes.

A newly isolated alkaline protease-producing myxobacterium was isolated from soil. The strain was identified as Pyxidicoccus sp. S252 on the basis of 16S rRNA sequence analysis. The extracellular alkaline proteases produced by isolate S252 (PyCP) was optimally active in the pH range of 11.0–12.0 and temperature range of 40–50°C The zymogram of PyCP showed six caseinolytic protease bands. The proteases were stable in the pH range of 8.0–10.0 and temperature range of 40–50°C. The activity of PyCP was enhanced in the presence of Na+, Mg2+, Cu2+, Tween-20, and hydrogen peroxide (H2O2) (hydrogen peroxide), whereas in Triton X-100, glycerol, ethylenediaminetetraacetic acid (EDTA), and Co2+, it was stable. PyCP showed a potential in various applications. The addition of PyCP in the commercial detergent enhanced the wash performance of the detergent by efficiently removing the stains of tomato ketchup and coffee. PyCP efficiently hydrolyzed the gelatin layer on X-ray film to release the embedded silver. PyCP also showed potent dehairing of goat skin and also efficiently deproteinized sea shell waste indicating its application in chitin extraction. Thus, the results of the present study indicate that Pyxidicoccus sp. S252 proteases have the potential to be used as an ecofriendly replacement of chemicals in several industrial processes.

Biochemical properties and repression studies of an alkaline serine protease from a haloalkaliphilic actinomycete, Nocardiopsis dassonvillei subsp. albirubida OK-14

A marine haloalkaliphilic actinomycete identified as Nocar diopsis dassonvillei subsp. albirubida (strain OK-14) was screened for its ability to produce extracellular alkaline protease under various physical conditions pertinent to different applications. The strain utilized amino acids as the sole source of nitrogen, displaying repression and de-repression phenomenon above the threshold levels. The activity of the partially purified alkaline protease was 1421.7 U/ml/min. The enzyme was optimally active at pH 10 and 60 °C. The activity was assessed under various concentrations of NaCl, organic solvents and metal ions. The partially purified alkaline protease was then examined for its suitability in blood de-staining, gelatine degradation and keratinase activity, implicating its relevance in detergent industries, gelatine extraction from X-ray films and degradation of bio-wastes. • Repression and de-repression of protease by different amino acids. • Protease stability in solvents, metal ions and various other agents. • Highly efficient in gelatin deproteinization and silver recovery. • Compatible as detergent supplement.

Enzymatic activity of alkaline protease from Bacillus cereus TD5B and its application as sheep skin dehairing agent

This study aims to determine the enzymatic activity of extracellular alkaline protease from Bacillus cereus TD5B and its potential application as a sheep skin dehairing agent. The B. cereus TD5B was screened for extracellular alkaline protease production on skim milk agar media, while its alkaline protease activity and the application were measured at 1%, 1.5%, and 2%. The application of alkaline protease from B. cereus TD5B as a sheep skin dehairing agent was observed through histological examination and physical properties measurement after chrome-tanning with lime and Na2S as control. The study was conducted in a completely randomized design, and the quantitative data were analyzed using Duncan’s Multiple Range Test. The results showed that a clear zone was seen surrounding B. cereus, indicating the bacteria’s proteolytic activity. The protease activity measurement showed that 2% of alkaline protease had the highest enzymatic activity at 144.75 U/mL/min. The highest tensile strength of sheep leather was obtained after dehairing at 1% alkaline protease concentration (350.26 kg/cm2), even though the highest elongation was obtained at 2% (34.92%). In contrast, different concentrations showed similar shrinkage temperatures at 90°C. This study concludes that the optimum alkaline protease concentration from Bacillus cereus TD5B as a sheep dehairing agent was 2%.

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.

AprD is important for extracellular proteolytic activity, physicochemical properties and spoilage potential in meat-borne Pseudomonas fragi

Pseudomonas fragi is a common spoilage agent of aerobically stored chilled meat. Its extracellular protease contributes to the spoilage of meat. AprD is considered an important component involved in the secretion of an alkaline extracellular protease in Pseudomonas. Thus, it is very possible that aprD plays an important role in spoilage regulation in P. fragi. The objective of the present study was to investigate the effects of aprD on bacterial proteolytic activity, biological properties, and in situ spoilage potential, as well as the mode of action of the protease AprA in spoiled meat. The results showed that aprD is important for surface-associated behaviors, as an ΔaprD mutant altered autoaggregation, swimming motility, and biofilm formation. aprD also contributes to the spoilage activities of P. fragi by affecting extracellular protease activity, total volatile basic nitrogen production, pH and the sensory traits of meat samples. These results highlight the presence of aprD-mediated regulation profiles in P. fragi and may provide new perspectives on chilled meat preservation.

Molecular Characterization of Alkaline Protease Gene Isolated from Aeromonas hydrophila strain AH10


 Alkaline protease enzymes are enzymes which can catalyze the process of proteolysis between the pH ranges from 8 to 12. Extracellular alkaline proteases are used as additives in detergent powders. In the present study, source of the organism was from a detergent contaminated area. The study has been carried out in Aeromonas hydrophila AH10 strain that produces protease enzyme with an alkaline pH optimum. The organism was a gram-negative rod with a protease enzyme activity of 0.385 ml/min. purification of the protease enzyme from the bacteria was carried out. This protease is suitable for use in alkaline detergent powders as well as in silver recovery process. The Aeromonas hydrophila strain AH10 gene encoding this high-alkaline protease was cloned and characterized. 
 Int. J. Appl. Sci. Biotechnol. Vol 8(2): 154-160

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.