High Alkaline Protease Research Articles

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.

Statistical optimization of thermostable alkaline protease from Bacillus cereus KM 05 using response surface methodology

Proteases have gained great attention due to their enormous applications in food, tannery, detergent, photography and many other industries. Proteases rank third position in the production of enzymes. This paper targets to isolate a bacterium with high alkaline protease activity and optimization of its production conditions using Response Surface Methodology (RSM). A bacterium isolated from soil contaminated with detergent exhibited clearance zone on skim milk agar medium with a protease activity of 22 U/ml. The bacterial strain was identified as Bacillus cereus KM05 and optimization of its production conditions were performed using statistical methods. Further optimization with Box Behnken design resulted in an increase in protease activity by 1.5-fold (28.6 U/ml). The protease enzyme was thermotolerant up to 70°C with stability towards alkaline pH (pH 9). The enzyme was not affected by most of the metal ions and solvents. Moreover, the protease was also compatible with six commercial detergents tested. Densitometric analysis of the destained fabric materials following the detergent-enzyme treatment, revealed a stain removal efficiency of 97%. The alkaline protease enzyme obtained was stable at different conditions with stain removal efficacy. Hence, the present alkaline protease could be used for detergent formulations.

Production, Purification, and Characterization of an Industrially Important Enzyme Alkaline Protease Produced from Locally Isolated <i>Bacillus</i> Bacteria

The worldwide demand for industrially essential enzymes is growing due to their eco-friendly and sustainable applications in broad industrial sectors. The main objective of this investigation is intended to isolate potent alkalophilic native bacteria from local habitats for the cost-effective production of an enzyme alkaline protease. For screening of alkalophilic bacteria, bacterial samples were taken from various natural alkaline habitats, and bacterial cultures were screened on agar plate having skimmed milk as protein substrate by using the protein hydrolysis method. The bacteria showing maximum proteolytic potential were identified by microscopic, biochemical, and 16S rDNA analysis. Additionally, the culture components and other medium parameters have been optimized for higher enzyme production. High yield alkaline protease was obtained with conditions of 1% inoculum, pH 9.0, and at 37°C temperature, with 1% sugarcane molasses as the best suitable carbon substrate. Partial pur¬ification of the enzyme was performed and characterized for their optimum activity on various parameters (temperatures and pH range). The approx. molecular weight of the enzyme was estimated to be ~35 kDa. This study demonstrates the production of an industrially important enzyme (alkaline protease) from a newly isolated native Bacillus spp. Economical production of this enzyme can be commercially applied in vast industrial sectors like agriculture, textile, food, detergent, and leather industries.

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

Pathogenicity of three entomopathogenic fungi, to the aphid species, Metopolophium dirhodum (Walker) (Hemiptera: Aphididae), and their Alkaline protease activities

The aim of the present study is to investigate the effect of three entomopathogenic fungi (EPF) (Beauveria bassiana, Cladosporium cladosporioides, and Verticillium alfalfae) on the aphid species, Metopolophium dirhodum (Walker) (Hemiptera: Aphididae). The selected EPF were isolated from the agricultural soil of the National Institute of Plant Protection (INPV) in Constantine, Algeria, and were tested against the aphid insects that were collected from the same area. The aphid species M. dirhodum were exposed to each fungal spore suspensions 107 conidia/ml for 10 s. Percent mortality was recorded at 1, 3, 5, and 7 days post treatment. Percentage mortalities, 7 days post treatment, were 95.83, 63.98, and 51.83% by B. bassiana, C. cladosporioides, and V. alfalfae, respectively. The higher protease activities were observed for isolate V. alfalfae with 95 U/ml, followed by B. bassiana with 38.26 U/ml, and finally C. cladosporioides with 35, 65 U/ml. The results presented in this study revealed that there was no relation between high alkaline protease activities and high virulence isolates.

Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2

A protease-producing strain CT2 isolated from Tunisian potatoes, exhibiting a potent protease activity (prot CT2), was identified as Bacillus halotolerans according to 16S ribosomal DNA sequence analysis. Maximum prot CT2 production was obtained in medium supplemented with bean seed proteins. Proteolytic activity was purified by ammonium sulphate precipitation, Sephacryl S-200 gel filtration and SP-sepharose cation-exchange chromatography. Optimal enzyme activity was reached at pH 9 and temperature of 50 °C. Proteolytic activity was enhanced by Ca2+ and Mn2+ ions, completely inhibited by PMSF suggesting a serine protease nature and exhibited high stability in the presence of commercial detergents. Prot CT2 showed broad substrate specificity towards both synthetic and natural substrates, with a high capacity to hydrolyze legume seed proteins. Using electrophoretic analysis, its molecular weight was around 250 kDa with two major subunit showing important homologies with serine proteases belonging to the subtilisin-like serine proteases. Based on the Lineweaver–Burk plots Km and Vmax values were 10 mg/ml and 50,000 U/mg respectively. This newly described prot CT2 displays relevant properties which highlight its potential use in various industrial and biotechnological applications.

Isolation and Characterization of Phosphate-Solubilizing Bacteria from Mushroom Residues and their Effect on Tomato Plant Growth Promotion.

Phosphorus is a major essential macronutrient for plant growth, and most of the phosphorus in soil remains in insoluble form. Highly efficient phosphate-solubilizing bacteria can be used to increase phosphorus in the plant rhizosphere. In this study, 13 isolates were obtained from waste mushroom residues, which were composed of cotton seed hulls, corn cob, biogas residues, and wood flour. NBRIP solid medium was used for isolation according to the dissolved phosphorus halo. Eight isolates produced indole acetic acid (61.5%), and six isolates produced siderophores (46.2%). Three highest phosphate-dissolving bacterial isolates, namely, M01, M04, and M11, were evaluated for their beneficial effects on the early growth of tomato plants (Solanum lycopersicum L. Wanza 15). Strains M01, M04, and M11 significantly increased the shoot dry weight by 30.5%, 32.6%, and 26.2%, and root dry weight by 27.1%, 33.1%, and 25.6%, respectively. Based on 16S rRNA gene sequence comparisons and phylogenetic positions, strains M01 and M04 belonged to the genus Acinetobacter, and strain M11 belonged to the genus Ochrobactrum. The findings suggest that waste mushroom residues are a potential resource of plant growth-promoting bacteria exhibiting satisfactory phosphate-solubilizing for sustainable agriculture.

Extracellular Proteome Profiling of Bacillus pumilus SCU11 Producing Alkaline Protease for Dehairing

Bacillus pumilus is one of the most characterized microorganisms that are used for high-level production of select industrial enzymes. A novel B. pumilus SCU11 strain possessing high alkaline protease activity was obtained in our previous work. The culture supernatant of this strain showed efficient dehairing capability with minimal collagen damage, indicating promising potential applications in the leather industry. In this study, the strain's extracellular proteome was identified by LC-MS/MS-based shotgun proteomic analysis, and their related secretory pathways were characterized by BLAST searches. A total of 513 proteins, including 100 actual secreted and 413 intracellular proteins, were detected in the extracellular proteome. The functions of these secreted proteins were elucidated and four complete secretory systems (Sec, Tat, Com, and ABC transporter) were proposed for B. pumilus. These data provide B. pumilus a comprehensive extracellular proteome profile, which is a valuable theoretical and applicative basis for future genetic modifications and development of industrial enzymes.

In silico mining for alkaline enzymes from metagenomic dna data of gut microbes of the lower termite Coptotermes gestroi in Vietnam

The high alkaline proteases, lipases, cellulases and hemicellulases are important enzymes in research and industries. In this study, using the Alcapred software, the metagenomic DNA sequences of the gut flora of Coptotermes gestroi were analyzed to identify the enzymes that were specifically adapted to alkaline condition. The results show that 737 of 943 ORFs (accounting for 72%) encoded proteases, 154 of 214 ORFs (holding 78%) encoded lipases and 338 of 575 ORFs (accounting for 59%) encoded cellulase and hemicelluase. All those enzymes were predicted to be alkaline enzymes. This study provide an overview picture of the alkaline enzyme groups of the gut flora of C. gestroi, and provide a good database for mining, isolation of the genes to produce recombinant enzymes.

Antagonistic Bacillus cereus TC-1 Isolated from Solar Salt Work in Southern India

Bacillus cereus TC-1 was isolated from condenser pond of manmade solar salt pan Thamaraikulam, Tamilnadu, India effectively suppressed the shrimp bacterial pathogens Vibrio harveyi, V. parahaemolyticus, V. anguillarum, V. alginolyticus and V. vulnificus by in vitro antagonistic assay of 9 to 15 mm of zone of inhibition. Phylogenetic analysis revealed that, B. cereus TC-1 was highly similar that the B. thuringiensis strain (100%) and other Bacillus sp. Their optimum growth between at the range of 4 to 6% of NaCl in the growth media and significantly (P<0.05) high alkaline protease production (170.85 U/ml) was observed in the NaCl of 6 %. Based on the antagonistic activity of the B. cereus TC-1 against the shrimp pathogens and its antimicrobial factors, it may be used as probiotics and developing novel antimicrobial bioactive substances against aquatic pathogens.Bangladesh J Microbiol, Volume 29, Number 1, June 2012, pp 27-31

Modulation of digestive and absorptive processes with age and/or after a dietary change in gilthead sea bream

During a 19-week trial, sea bream juveniles were fed with two isonitrogenous and isoenergetic diets in which 75% of the fish meal was replaced by plant protein sources and that only differed in the lipid source: fish oil (FO) or vegetable oil (VO). After the growth period, intestinal pH content, digestive enzyme activities, nutrient absorption capacity and final weight were studied in the pre-feeding stage on the FO or VO diet and also after a dietary change at different animal weights (92g, 147g, 233g), both in the short term and at the end of the trial.Intestinal pH content, alkaline protease, α-amylase and lipase activities and nutrient absorption capacities decreased as the animals grew. Moreover, the VO group tended to have higher alkaline protease and α-amylase activities, whereas lipase activity was higher in the FO group; these changes were significant in small fish. The absorption capacities of essential amino acids were higher than those of non-essential amino acids and d-Glc. Moreover, absorption capacities were higher in small VO animals compared to FO. No changes in growth rate were found at the end of the trial. However, FO animals had a higher growth rate during the first half of the trial after which growth slowed, whereas VO animals grew at a lower but more steady rate throughout the trial.Small animals were not able to adapt their digestive enzyme activities or nutrient absorption capacities to the new or original feeding condition after a short-term dietary change, whereas large animals generally adapted.After 9–15weeks on the FO diet, animals were able to adapt their protease activity and absorption capacity to the new situation; however, α-amylase activity declined. In contrast, after changing to the VO diet animals up-regulated their nutrient absorption capacities and α-amylase activity whereas alkaline protease activity tended to decrease. At the end of the trial final body weight was not affected by dietary treatment. Statement of relevance“Wash out” or FO diet feeding in early stages and VO later?

Identification and characterization of alkaline protease producing Bacillus firmus species EMBS023 by 16S rRNA gene sequencing.

Probiotic microorganisms are those which exert a positive exect on the growth of the host, when administered as a dietary mixture in an adequate amount. They form the best alternative to the use of antibiotics for controlling enteric diseases in poultry farm animals, especially in the light of the gruesome problems of development of antibiotic resistance in enteric pathogens and the contamination of poultry products with antibiotics. 16S rDNA sequencing which has gained wide popularity amongst microbiologists for the molecular characterization and identification of newly discovered isolates provides accurate identification of isolates down to the level of sub-species (strain). It's most important advantage over the traditional biochemical characterization methods are that it can provide an accurate identification of strains with atypical phenotypic characters as well. The following work is an application of 16S rRNA gene sequencing approach to identify a novel, alkaline protease producing bacteria, from poultry farm waste. The sample was collected from a local poultry farm in the Guntur district, Andhra Pradesh, India. Subsequently the sample was serially diluted and the aliquots were incubated for a suitable time period following which the suspected colony was subjected to 16S rDNA sequencing. The results showed the isolate to be a novel, high alkaline protease producing bacteria, which was named Bacillus firmus isolate EMBS023, after characterization the sequence of isolate was deposited in GenBank with accession number JN990980.

Enhanced thermostability of a fungal alkaline protease by different additives.

A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory.

Secretory Expression, Functional Characterization, and Molecular Genetic Analysis of Novel Halo-Solvent-Tolerant Protease from Bacillus gibsonii

A novel protease gene from Bacillus gibsonii, aprBG, was cloned, expressed in B. subtilis, and characterized. High-level expression of aprBG was achieved in the recombinant strain when a junction was present between the promoter and the target gene. The purified recombinant enzyme exhibited similar N-terminal sequences and catalytic properties to the native enzyme, including high affinity and hydrolytic efficiency toward various substrates and a superior performance when exposed to various metal ions, surfactants, oxidants, and commercial detergents. AprBG was remarkably stable in 50% organic solvents and retained 100% activity and stability in 0-4 M NaCl, which is better than the characteristics of previously reported proteases. AprBG was most closely related to the high-alkaline proteases of the subtilisin family with a 57-68% identity. The secretion and maturation mechanism of AprBG was dependent on the enzyme activity, as analyzed by site-directed mutagenesis. Thus, when taken together, the results revealed that the halo-solvent-tolerant protease AprBG displays significant activity and stability under various extreme conditions, indicating its potential for use in many biotechnology applications.

Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation

A protease- and chitosanase-producing strain was isolated and identified as Bacillus cereus TKU022. The protease and chitosanase were both produced using 1.5% (w/v) shrimp head powder (SHP) as the sole carbon/nitrogen source, and these enzymes were purified from the culture supernatant. The molecular masses of the TKU022 protease and chitosanase determined using SDS–PAGE were approximately 45 and 44kDa, respectively. The high stability of the TKU022 protease toward surfactants, an optimal pH of 10 and an optimal temperature of 50–60°C suggest that this high-alkaline protease has potential applications for various industrial processes. Concomitant with the production of the TKU022 chitosanase, N-acetyl chitooligosaccharides were also observed in the culture supernatant, including (GlcNAc)2, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 at concentrations of 201.5, 12.4, 0.5, and 0.3μg/mL, respectively, as determined using an HPLC analysis. The chitin oligosaccharides products were also characterized using a MALDI-TOF mass spectrometer. A combination of the HPLC and MALDI-TOF MS results showed that the chitin oligosaccharides of the TKU022 culture supernatant comprise oligomers with degree of polymerization (DP) from 2 to 6. Using this method, the production of a protease, a chitosanase, and chitin oligosaccharides may be useful for various industrial and biological applications.

English

Development of natural antimicrobial strategies has always attracted researchers, as disease outbreaks are recognized as im- portant constraints to living organisms because of the development of antibiotic resistance in microorganisms. Use of probiotic bacteria has been in practice as alternatives to antimicrobials in disease control as microbial control agents. Probiotics, the natural and beneficial micro flora has been widely accepted and used in farming and aquaculture because of its diversified applications. It improves water and pond sedi- ment quality which inturn improves aquatic life. Bacterial pathogenicity and virulence can be controlled without using excessive antibiotics which help to minimize the risk of multiple antibiotic resistances. Probiotic help in increased productivity and profits when used properly as they produce antagonistic compounds that are inhibitory toward pathogens, compete with harmful microorganisms for nutrients and energy, compete with deleterious species for adhesion sites, enhance the immune response of the animal, improve water quality, and interact with phytoplankton. Among the large number of probiotic products in use today are bacterial spore formers, mostly of the genus Bacillus. The genus Bacillus is widely used as probiotic products because of its extremely resistant spores which provide exceptional longevity and re- lease exoenzymes. The current investigation is to identify a novel strain of Bacillus licheniformis by application of 16S rRNA gene sequenc- ing. The approach is to identify a novel, proteases and amylases producing bacteria which is used for detergent production. The sample was isolated from near Gudiwada, Krishna district, Andhra Pradesh, India. Subsequently the sample was serially diluted and the aliquots were incubated for a suitable time period following which the suspected colony was subjected to 16S rDNA sequencing. The results showed the isolate to be a novel, high alkaline protease producing bacteria, which was named Bacillus licheniformis strain EMBS026, after characteriza- tion the sequence of isolate was deposited in GenBank with accession number JQ267798. Key words- 16s Gene sequencing, Bacillus licheniformis strain EMBS026, Proteases and amylases producing bacteria.

High-level expression of alkaline protease using recombinant Bacillus amyloliquefaciens

Bacillus licheniformis CICIM B5102 was used for the commercial production of alkaline protease. The full-length gene apr encoding the alkaline protease was amplified via polymerase chain reaction (PCR) using the genomic DNA of B. licheniformis CICIM B5102 as a template. The apr gene was cloned into plasmid pUB110, resulting in the recombinant plasmid pUB-apr, which was then transformed into Bacillus amyloliquefaciens CICIM B4803. The protease productivity was significantly improved in the transformants of B. amyloliquefaciens CICIM B4803. A transformant with high alkaline protease productivity was selected, and the alkaline protease productivity of the strain increased by 46% when compared with that of wild-type B. licheniformis CICIM B5102. Key word: Alkaline protease, Bacillus amyloliquefaciens, Bacillus licheniformis.

Characterization of a Novel Surfactant and Organic Solvent Stable High-alkaline Protease from New Bacillus pseudofirmus SVB1

Characterization of a Novel Surfactant and Organic Solvent Stable High-alkaline Protease from New Bacillus pseudofirmus SVB1

Molecular Cloning, Sequence and Structural Analysis of Dehairing Mn2+ Dependent Alkaline Serine Protease (MASPT) of Bacillus pumilus TMS55

Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine β-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.

Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family

High-alkaline proteases are of great importance because of their proteolytic activity and stability under high-alkaline condition. We have previously isolated a new protease (AprB) which has potential industrial applications based on its high-alkaline adaptation. However, the molecular and structural basis for alkaline adaptation of this enzyme has not been fully elucidated. In the present study, AprB gene was cloned and expressed in the Bacillus subtilis WB600. This gene codes for a protein of 375 amino acids comprised with a 28-residual signal peptide, a 78-residual pro-peptide, and a 269-residual mature protein. The deduced amino acid sequence has the highest homology of 63.2% with that of the high-alkaline proteases. Recombinant AprB was purified and determined to be monomeric with molecular mass of 26.755 kDa. The NH 2-terminal sequence of the purified AprB was A-Q-S-I-P-W-G-I-E-R. This enzyme exhibited high catalytic efficiencies ( K cat/ K m) towards natural, modified, and synthesis substrates with optimal activity at 60 °C and pH 10. AprB was stable over a wide range of pH 5 to 11 and various surfactants, and could be activated by Mg 2+, Ca 2+ and Ba 2+. The structural properties of AprB, like a higher ratio of R/( R + K), a larger area of hydrophobic surface, increased number of ion pairs formed by Arg residue, and the exposure of Asp active residue on the surface, might be responsible for its alkaline adaptation. In contrast with members of subtilisin family, such as M-protease and subtilisin BPN′, AprB harbored a high content of Glu and Asp residues, and a low content of Arg and Lys residues on the surface. Interestingly, these structural characters were similar with that of psychrophilic proteases, which suggested that these molecular factors were not restricted in the psychrophilic proteases, and therefore were not solely responsible for their cold-adaptation. Our results reveal a novel structural feature of AprB unique to subtilisin family and provide clues for its alkaline adaptation.