Extracellular Alkaline Protease Research Articles

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.

Spo0A can efficiently enhance the expression of the alkaline protease gene aprE in Bacillus licheniformis by specifically binding to its regulatory region

The expression of enzymes in Bacillus licheniformis, such as the valuable extracellular alkaline protease AprE, is highly regulated by a complex transcriptional regulation mechanism. Here, we found that the transcript abundance of aprE varies >343-fold in response to the supply of nutrients or to environmental challenges. To identify the underlying regulatory mechanism, the core promoter of aprE and several important upstream regulatory regions outside the promoter were firstly confirmed by 5′-RACE and mutagenesis experiments. The specific proteins that bind to the identified sequences were subsequently captured by DNA pull-down experiments, which yielded the transcriptional factors (TFs) Spo0A, CggR, FruR, YhcZ, as well as fragments of functionally unassigned proteins. Further electrophoretic mobility shift assay (EMSA) and DNase I foot-printing experiments indicated that Spo0A can directly bind to the region from −92 to −118 nucleotides upstream of the transcription start site, and the deletion of this specific region drastically decreased the production of AprE. Taken together, these results indicated that the expression of aprE was mainly regulated by the interplay between Spo0A and its cognate DNA sequence, which was successfully applied to overproduce AprE in a genetically modified host harboring three aprE expression cassettes. The DNA binding proteins may serve to increase the efficiency of transcription by creating an additional binding site for RNA polymerase. The discovery of this mechanism significantly increases our understanding of the aprE transcription mechanism, which is of great importance for AprE overproduction.

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangroves

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangroves

RNA sequencing reveals small RNAs in Bacillus pumilus under different growth phases of the protease fermentation process.

Bacillus pumilus, an endospore-forming soil bacterium, produces a wide array of extracellular proteins, such as proteases, which are already applied in the chemical, detergent and leather industries. Small noncoding regulatory RNAs (sRNAs) in bacteria are important RNA regulators that act in response to various environmental signals. Here, an RNA-seq-based transcriptome analysis was applied to B. pumilus SCU11, a strain that produces extracellular alkaline protease, across various growth phases of the protease fermentation process. Through bioinformatics screening of the sequencing data and visual inspection, 84 putative regulatory sRNAs were identified in B. pumilus, including 21 antisense sRNAs and 63 sRNAs in intergenic regions. We experimentally validated the expression of 48 intergenic sRNAs by quantitative RT-PCR (qRT-PCR). Meanwhile, the expression of 6 novel sRNAs was confirmed by northern blotting, and the expression profiles of 5 sRNAs showed close correlation with the growth phase. We revealed that the sRNA Bpsr137 was involved in flagellum and biofilm formation in B. pumilus. The identification of a global set of sRNAs increases the inventory of regulatory sRNAs in Bacillus and implies the important regulatory roles of sRNA in B. pumilus. These findings will contribute another dimension to the optimization of crucial metabolic activities of B. pumilus during a productive fermentation process.

Characterization and fermentation optimization of novel thermo stable alkaline protease from Streptomyces sp. Al-Dhabi-82 from the Saudi Arabian environment for eco-friendly and industrial applications

In this study a novel thermo stable alkaline protease producing Streptomyces sp. Al-Dhabi-82was isolated from the soil. The effect of fermentation period on enzyme production by Streptomyces sp. Al-Dhabi-82 was optimized in submerged fermentation. Protease activity was found to be maximum after 5 days of incubation (129.5 ± 7.1 U/ml) and depleted after 6 days of incubation (113.8 ± 4.1 U/ml). Enzyme production increases with the increase in pH up to 9.0 (136.2 ± 3.6 U/ml) and enzyme production depleted significantly at pH 11.0 (67 ± 2.9 U/ml). Maximum production of protease was observed at 40 °C (164 ± 11.1 U/ml). Among the evaluated carbon sources, maltose significantly influenced on protease production (212 ± 14.8 U/ml). The optimum amount of protease (269 ± 10.4 U/ml) produced by Streptomyces sp. Al-Dhabi-82 was observed in the production medium containing yeast extract. Enzyme production was maximum in the presence of 0.15% Ca2+. The specific activity of crude enzyme was 26 U/ mg protein and it increased as 276 U/mg protein after chromatography separation. The molecular weight of purified protease obtained from sephadex G-75 gel filtration chromatography was estimated to be 37 kDa using SDS-PAGE. This enzyme showed high activity at pH 9.0, and lost about 16% activity at pH 10. The optimum temperature for Streptomyces sp. Al-Dhabi-82 protease was 40 °C. The extracellular alkaline protease from Streptomyces sp. Al-Dhabi-82 hydrolyzed chicken feather completely.

Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive

A thermostable extracellular alkaline protease (called SAPA) was produced (4600U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70°C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.

Purification and biochemical characterization of a novel thermostable and halotolerant subtilisin SAPN, a serine protease from Melghiribacillus thermohalophilus Nari2AT for chitin extraction from crab and shrimp shell by-products.

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2AT. The highest yield of protease (395IU/g) with white shrimp shell by-product (40g/L) as a unique source of nutriments in the growth medium was achieved after 52h at 55°C. The monomeric enzyme of about 30kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75°C, respectively, and half lives of 9 and 5h at 80 and 90°C, respectively. The sequence of the 25 NH2-terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products.

Molecular characterization of alkaline protease-coding gene from Bacillus licheniformis MK90 mutants with biofilm inhibitory activity

Background Enzymes are organic materials that accelerate biochemical processes without themselves undergoing change. They can be produced by plant, animal, fungi, and bacteria. Bacterial proteases are much favorable than any other sources, because bacteria can grow quickly and can be easily cultivated in laboratory. Objective To isolate and screen bacteria from soil samples for their ability to produce alkaline protease, and to improve the alkaline protease production followed by evaluation of its antimicrobials and antibiofilm activity. Materials and methods Sample collection was carried out from different locations in Egypt. Isolation of bacteria from soil samples was done using serial dilution method on skim milk agar. All isolated bacteria were screened for their ability to produce protease enzyme. The bacterial isolate showing maximum alkaline protease activity was identified using 16 S rRNA genetic identification. To induce mutations, ultraviolet (UV) irradiation was used. The most active mutant strains were selected for production, purification, and characterization of alkaline protease followed by evaluation of alkaline protease antimicrobial and antibiofilm activity. Results and conclusion Three UV mutants (MT2, MT4, and MT26) out of 48 displayed proteolytic activity more than other mutants and wild type (WT). Bacillus alkaline extracellular protease gene was genetically characterized through isolation of the genomic DNA of Bacillus licheniformis MK90 WT, and the best protease-producing UV mutants were followed by amplification, sequencing, and analyses. WT strain and best protease-producing mutants were compared at proteomic level through sodium dodecyl sulfate polyacrylamide gel electrophoresis for total cellular proteins. Then protease enzyme of WT and mutants was purified and characterized. This study reports that the B. licheniformis protease was active at an alkaline pH and wide range of temperatures (40–60°C), reflecting its potential application in detergent and laundry industries. On the contrary, the antibiofilm activity of the protease enzymes was evaluated toward four pathogenic bacterial strains, i.e., Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis, and the results showed that proteases from B. licheniformis MK90 may be useful for controlling biofilm formation by some pathogenic bacteria.

Heterologous Expression of Histidine Acid Phytase from Pantoea sp. 3.5.1 in Yarrowia lipolytica

The dimorphic yeasts Yarrowia lipolytica are used as effective expression system and are characterized by a high level of production of heterologous protein. In this work, we aimed to clone and express Pantoea sp. 3.5.1 agpP phytase gene in Yarrowia lipolytica. Genetic constructs containing the native phytase gene (agpP) under the control of the strong hybrid promoter hp4d and the signal peptide of the alkaline extracellular protease XPR2 gene, as well as the optimized gene (agpP-opt) of phytase under the control of two signal peptides—bacterial and yeast were obtained. Recombinant Y. lipolytica strains with integrated bacterial phytase genes were obtained and expression of phytase was analyzed.

Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes

Alkaline proteases have applications in numerous industries. In this study, we have isolated and screened proteolytic bacteria from poultry wastes mixed soil and identified two bacterial isolates as Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 based on 16S rDNA sequencing. Maximum level of protease production was achieved after 24 h of fermentation in a basal medium. The optimal temperature, initial pH of the media and agitation for alkaline protease production by these two isolates were 30 °C, pH 9.0 and 120 rpm, respectively. The both bacterial isolates produced maximum level of protease with 3.0% organic municipal solid wastes (OMSW) as the sole source of carbon and nitrogen under previously optimized fermentation conditions. In comparison with the shake flask, protease production increased about 2.5-fold in the bioreactor with reduction in fermentation period. The partial purification of protease resulted in a final 45.67 and 34.86-fold purified protease with a specific activity of 8335.34 and 9918.91 U/mg protein and a typical yield of 9.75 and 9.41% from B. subtilis and E. indicum, respectively. The optimum temperature and pH of the partially purified protease from the both sources was 40 °C and pH 9.0, respectively. Protease from the both isolates was stable at pH 7.0–12.0 and at temperatures up to 50 °C. The effects of protease inhibitors indicated that the protease from B. subtilis might be serine and cysteine type and from E. indicum might be cysteine type. Mg2+, K+ and Ca2+ stimulated but Zn2+, Hg2+, Co2+ and Fe3+ strongly inhibited the protease activity. The partially purified protease from B. subtilis substantially dehaired cow skin and decomposed gelatinous compound from X-ray film. Our study revealed that OMSW can be used as raw material for production of bacterial extracellular protease and alkaline protease from B. subtilis might be potential for industrial and biotechnological applications.

Characterization of thermoadaptive serine metalloprotease and application in waste management

Thermoadaptive enzymes offer new opportunities for bio-catalysis and bio-transformations owing to their ability to function over wide temperature ranges. Microbial alkaline proteases are important constituents of commercial enzyme preparations, accounting for more than 60% of the total industrial enzyme market. Fibrous proteins, such as horn, feathers etc. are abundantly available in nature as recalcitrant solid wastes. Extracellular alkaline proteases derived from microorganisms can be used for degradation of proteinaceous wastes and convert them into value-added products e.g. peptide concentrates or amino acids. The present study relates to the isolation of an extracellular, bacterial thermoadaptive serine metalloprotease from the soil. The purified protease showed a remarkable increase in thermostability (4 °C–100 °C) that was enhanced in the presence of Ca2+. The protease (+Ca2+) was utilized to efficiently degrade fish scales and feather to produce amino acids, as declogging agent and for removing blood stains from cloth with a short reaction time. The ability of the protease could be utilized in bulk for solid waste management providing for an eco-friendly and effective process.

Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A

A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 °C) followed by ammonium sulfate precipitation (40–70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH2-terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 °C. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference.

Thermodynamics of a Ca2+ dependent, highly thermostable and detergent compatible purified alkaline serine protease from Nocardiopsis xinjiangensis strain OM-6

Extracellular alkaline protease producing salt tolerant alkaliphilic actinobacteria, Nocardiopsis xinjiangensis strain OM-6 was isolated from Okha Madi (OM) site of coastal Gujarat, India. The purified protease was stable even at 70°C in 100mM Ca2+ with Kd=20×10−3 and t1/2=34min. The activation energies (E), enthalpy (∆H*) and entropy (∆S*) for protease deactivation were 29.35kJ/mol, 26.68kJ/mol and −186.22J/mol, respectively in 200mM Ca2+. The ∆G* for protease deactivation was 97.63kJ/mol at 50°C in 100mM Ca2+. OM-6 protease exhibited enhanced residual activities up to 103%, 70%, 144% and 119% with SDS, CTAB, Tween 80 and Triton X-100, respectively after 2h of incubation at 40°C. Interestingly, residual activity of OM-6 protease increased by 450% and 559% in 50mM H2O2 and 10mM β-mercaptoethanol respectively even after 2h of incubation. Moreover, protease retained 100% of its original activity with H2O2 and β-mercaptoethanol at highest concentration after 24h. The protease retained more than 60% of original activity with 1% w/v of each commercial detergent even after 2h at 40°C. These unique properties of protease make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.

Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangrovesM. Parthasarathy, J. Joel Gnanadoss

English

A Gram-positive, rod-shaped, spore-forming haloalkaliphilic bacterium designated as NA7 was isolated from the surface of a Helianthemum nummularium root sample obtained from Wadi Natrun in Egypt. Sequence analysis of the 16S rRNA gene revealed a Bacillus haloalkaliphilius strain as the closest match with 99% identity. In a shake flask culture containing 10% NaCl, adjusted to pH 10 and incubated at 37°C, the isolated strain produced thermostable extracellular alkaline protease with relatively stable maximum activity records (0.610-0.625 TU) within a relatively long stationary phase that exceeded 60 h. A 2-level fractional factorial design (Plackett-Burman) was then applied to screen for nutritional and cultivation factors regulating protease production by the isolate and to appraise their effects. Calculated statistical parameters revealed that NaCl and MgSO4 are the most significant independent variables affecting alkaline protease production by NA7 and suggested a near-optimum culture condition. Verification of this predicted condition resulted in an alkaline protease specific activity record of 509 TU/mg protein with a 1.27 fold increase when compared to the basal medium culture. Key words: Alkaline protease, Bacillus haloalkaliphilus, Wadi Natrun, haloalkaliphiles.

Eco-Friendly Applications of Bacterial Extracellular Alkaline Protease

Eco-Friendly Applications of Bacterial Extracellular Alkaline Protease

Characterization of the Crude Alkaline Extracellular Protease of Yarrowia lipolytica YlTun15

Yarrowia lipolytica YlTun15 (GeneBank Acc. N° MF327143), isolated from farmed Dicentrarchus labrax’s gills, secretes an alkaline extracellular protease, which exhibited the highest activity at pH 9 and temperature 45°C. The enzyme activity extracted was tested in the presence of different ion metal and protein inhibitors. The enzyme activity increased in the presence of both Cu2+ (1 mM) and Mn2+ (5mM) in the medium. K+, Na2+, Mg2+ and Ca2+ had no effect on the enzyme activity while Ni+, Hg+, Zn2+ and Fe2+ decreased significantly its relative activity to 43.63%, 66.25%, 30.75% and 19.48% respectively at the 5 mM level. The enzyme was almost (activity=1.47%) inhibited by phenylmethylsulfonyl fluoride at the concentration of 5 mM. However, the protease activity was relatively constant in the presence of EDTA and SDS that may conclude that this enzyme was not a metalloprotease and belong to the serine protease category. After 18 months-storage at -20°C, the enzyme activity has decreased to 23.17%. This protease may have a potential application in food and detergent activity.

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.

Alkaline proteases from a newly isolated Micromonospora chaiyaphumensis S103: Characterization and application as a detergent additive and for chitin extraction from shrimp shell waste

The present study was undertaken to characterize the extracellular thermostable serine alkaline proteases from newly actinomycete strain Micromonospora chaiyaphumensis S103 and to describe their evaluation in commercial detergents and shrimp waste deproteinization. This proteolytic crude extract was active and stable in alkaline solution. It was extremely stable in the pH range of 5.0–12.0. The optimum pH and temperature were 8.0 and 70°C, respectively, using casein as a substrate. The thermoactivity and thermostability of proteases were enhanced by the addition of 5mM Ca2+. Proteases from S103 were also used for shrimp wastes deproteinization in the process of chitin preparation. The percent of protein removal after 3h hydrolysis at 45°C with an enzyme/substrate ratio of 20U/mg had reached 93%. Furthermore, S103 crude enzyme was stable towards several organic solvents and retained 100% of its original activity after 90days of incubation in the presence of methanol, hexane, acetone, and DMSO. These properties make S103 proteases an ideal choice for application in detergent formulations, chitin production, and enzymatic peptide synthesis.