Alkaline Protease Gene Research Articles

Genomic insights and Taguchi-based optimization of culture conditions for enhanced alkaline protease production in Streptomyces barkulensis RC1831

This study presents a genome analysis identifying various alkaline protease genes and culture condition optimization to enhance the production of the enzyme in Streptomyces barkulensis RC1831. The analysis of genes using bioinformatics tools such as RAST, KEGG, KAAS, and BLASTx revealed the presence of 10 genes that are responsible for alkaline protease production belonging to different families. Multiple sequence alignment showed a high sequence identity with M24 family serine protease followed by subtilisin-like serine protease. Optimal conditions for enhanced protease production were determined to be at 37°C, pH 11, casein1 % (W/V), dextrose 0.5 % (W/V), urea 0.5 % (W/V), tryptophan 1 % (W/V), 1 mM Mn+2, 1 % (V/V) Tween-80 in LB medium and an incubation time of 72 hours. Out of the 10 alkaline protease genes, 2 genes expressed significantly according to the activity observed during optimization processes i.e., M24 family, Mn+2 dependent metalloprotease and subtilisin-like serine, ca+2 dependent metalloprotease. Furthermore, the AP enzyme was remarkably stable in the presence of various cofactors (Metal ions) and surfactants, indicating its potential for industrial applications under diverse conditions.

Exploring alkaline serine protease production and characterization in proteolytic bacteria Stenotrophomonas maltophilia: Insights from real-time PCR and fermentation techniques

This study focuses on identifying a potent microbe for efficient alkaline protease production, characterizing its properties, and investigating protease gene expression across diverse culture conditions. Stenotrophomonas maltophilia strain GEBN5 is methodically chosen and refined for peak alkaline protease production at 36 h (75.37 U/mL) in basal media employing precise adjustments in temperature, pH, incubation time, and carbon/nitrogen sources, achieving a noteworthy maximum protease activity of 97.03 U/mL in shake flask culture. Alkaline protease gene (StmPr1) up-regulation was explored via Real-time PCR under varied culture conditions (pH, temperature, carbon, and nitrogen sources) in a 2L fermenter at 150 rpm, resulting in peak protease production of 113 ± 0.53 U/mL in batch culture at pH 9.0, 35 °C, with sucrose and casein, respectively. The characterization data demonstrates exceptional stability of the current protease across a broad pH (9.0–11.0) and optimum temperature of 35 °C, highlighting its promising applications in diverse industrial and biotechnological settings. A higher instability complex of protein 28.59 was revealed by in silico analysis, indicating prolonged stability during hydrolysis and a wide range of potential uses. The purified protease demonstrates superior compatibility (45%–88% residual activity) with diverse detergent brands treated for 45 min at 40 °C, indicating its potential as a valuable additive for enhanced washing efficacy. Current research offers a novel strategy to boost protease production, presenting a promising alternative to chemical dehairing in leather industries and solid organic waste treatment. This approach holds the potential for mitigating environmental pollution through bioremediation.

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.

Genetic engineering for enhanced production of a novel alkaline protease BSP-1 in Bacillus amyloliquefaciens.

Alkaline protease has been widely applied in food, medicine, environmental protection and other industrial fields. However, the current activity and yield of alkaline protease cannot meet the demand. Therefore, it is important to identify new alkaline proteases with high activity. In this study, we cloned a potential alkaline protease gene bsp-1 from a Bacillus subtilis strain isolated in our laboratory. BSP-1 shows the highest sequence similarity to subtilisin NAT (S51909) from B. subtilis natto. Then, we expressed BSP-1 in Bacillus amyloliquefaciens BAX-9 and analyzed the protein expression level under a collection of promoters. The results show that the P43 promoter resulted in the highest transcription level, protein level and enzyme activity. Finally, we obtained a maximum activity of 524.12 U/mL using the P43 promoter after fermentation medium optimization. In conclusion, this study identified an alkaline protease gene bsp-1 from B. subtilis and provided a new method for high-efficiency alkaline protease expression in B. amyloliquefaciens.

Conjugational Transformation of Wild Type Bacillus halodurans CM1 by Methylated Recombinant Plasmid Harbouring a Gene Encoding for Alkaline Protease and the Protease Activity Assay of the Transformant

Protease contributes significantly to various industrial sectors, as shown by its increasing demand. An indigenous previously isolated bacteria, Bacillus halodurans CM1, is a wild-type bacterium whose ability to produce alkalotermophilic protease. In this study, the transformation of methylated pBBRE194 plasmid containing alkaline protease gene from this strain (PBBRE194-prot-CM1) into itself using a conjugation approach was conducted, and the measurement of the alkaline protease activity of the recombinant bacterium was carried out. The recombinant B. halodurans CM1 has been verified to carry the methylated pBBRE194 prot-CM1 by examining its ability to degrade protein in media containing skim milk and tetracycline, but also by amplifying tetracycline resistance gene sequence with 1,024 bp length of plasmid pBBRE194 prot-CM1 by PCR method from the recombinant bacterium. The results confirmed that recombinant B. halodurans CM1 was positively harboring plasmid pBBRE194 prot-CM1. Alkaline protease produced by recombinant CM1 reached higher activity than wild type between 18-36 h of cultivation. The alkalotermophilic wild type B. halodurans could accept the recombinant plasmid into their cells via conjugational transformation is firstly reported to our further knowledge.

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.

Formation of recombinant bifunctional fusion protein: A newer approach to combine the activities of two enzymes in a single protein.

The tissue of insects, pests, and fungi has a chitin layer followed by protein in the cell membrane. The complete biodegradation of chitin and protein-present in the waste requires the action of two enzymes, namely chitinase, and protease. Combining chitinase and protease in a single protein/enzyme will serve as a bifunctional enzyme that can efficiently degrade the chitin and protein-rich biomass. The present study was aimed to fuse these two enzymes to produce a single protein and study the kinetics of the recombinant fusion protein. A chitinase and alkaline protease genes were isolated, cloned, and expressed successfully as a fusion product in heterologous host Escherichia coli. The two native genes were successfully fused in E.coli by using flexible glycine-serine (G4S)2 linker (GGGGS, GS linker). The recombinant fusion protein in E.coli showed hydrolyzed chitin and protein on chitin and bovine serum albumin agar plates confirming the successful cloning and expression of chitinase and protease enzymes in a single fusion protein. The common pUC18-T7 mini vector with the ompA signal sequence helps the extracellular expression of fusion protein efficiently. The native gel electrophoresis revealed a molecular mass of purified protein as 92.0 kDa. The fusion protein's maximal chitinase and protease activity occurred at pH 5.0 and 8.0 and 30 0C, respectively resembling the individual enzymes'. In the kinetic studies of the fusion protein, it was observed that the presence of metal ions such as Cu2+, Na2+, and Ca2+; significantly enhanced the enzyme activities while organic solvents oxidants and chemicals have drastically affected the activities of both the enzymes in the fusion protein. No such fusion protein has been produced in a heterologous host yet. The reports on fusion protein with biomass-degrading capacity are also scarce. This is probably the first report of a bifunctional chitinase/protease expressed in E. coli.

Resistance to Aflatoxin Accumulation in Maize Mediated by Host-Induced Silencing of the Aspergillus flavus Alkaline Protease (alk) Gene.

Aspergillus flavus is a fungal pathogen that infects maize and produces aflatoxins. Host-Induced Gene Silencing (HIGS) has been shown to reduce host infection by various fungal pathogens. Here, the A. flavus alkaline protease (alk) gene was targeted for silencing through HIGS. An RNAi vector carrying a portion of the alk gene was incorporated into the B104 maize genome. Four out of eight transformation events containing the alk gene, Alk-3, Alk-4, Alk-7 and Alk-9, were self-pollinated to T4/T6 generations. At T3, the Alk-transgenic lines showed up to 87% reduction in aflatoxin accumulation under laboratory conditions. T4 transgenic Alk-3 and Alk-7 lines, and T5 and T6 Alk-4 and Alk-9 showed an average of 84% reduction in aflatoxin accumulation compared to their null controls under field inoculations (p < 0.05). F1 hybrids of three elite maize inbred lines and the transgenic lines also showed significant improvement in aflatoxin resistance (p < 0.006 to p < 0.045). Reduced A. flavus growth and levels of fungal ß-tubulin DNA were observed in transgenic kernels during in vitro inoculation. Alk-4 transgenic leaf and immature kernel tissues also contained about 1000-fold higher levels of alk-specific small RNAs compared to null controls, indicating that the enhanced aflatoxin resistance in the transgenic maize kernels is due to suppression of A. flavus infection through HIGS of alk gene.

Inhibitory effect of Saccharomyces cerevisiae supernatant and lysate on expression of lasB and apl genes of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen. Elastase and alkaline protease are two major virulence factors in this bacterium. Today, probiotics is one of the most comprehensive way to treat and prevent diseases. The aim of this study was the evaluation the effect of Saccharomyces cerevisiae S3 on the expression of elastase (lasB) and alkaline protease (apl) genes. In the first stage, S. cerevisiae S3 supernatant and lysate were prepared. Then, subminimum inhibitory concentrations (sub-MIC) of both supernatant and lysate were applied to P. aeruginosa PAO1. The expression of lasB and apl were measured by real-time PCR method. The supernatant of the probiotic decreased the apl gene expression and increased expression of lasB. The lysate extract decreased the expression of lasB and apl. The results of this study showed that S. cerevisiae S3 had an inhibitory effect on some virulence factors of P. aeruginosa. Further experiments on the influence of S. cerevisiae on other virulence factors of P. aeruginosa and pathogens are suggested.

Expression, purification, and molecular characterization of a full-length thermostable alkaline protease gene from Bacillus subtilis DMA-09

The present study aim was to isolate bacterial strains, producing protease, from soil samples of different industrial wastes and dumping areas of slaughter houses. For this, 25 bacterial isolates forming clear zones in skimmed milk agar plates as a result of casein hydrolysis were selected. One bacterial isolate showing the highest protease activity (400 U/ml) was identified as Bacillus subtilis through 16S rRNA gene sequencing. Full-length alkaline protease gene was amplified, sequenced, cloned in expression vector pT7-7, and transformed in E. coli BL21C. Over-expression of protease gene was determined in isopropyl β-d-1-thiogalactopyranoside (0.5 mM) at 37 °C for 3 h. Expressed alkaline protease was purified and 10 folds increased enzyme activity was determined as compared to the wild type B. subtilis DMA-09. The purified protease was detected on SDS-PAGE as a single band of 31 kDa and was stable at 90 °C and 85% enzyme activity was retained at 100 °C. The optimum enzyme activity was obtained at pH 10 and was enhanced in the presence of 5 mM each Mg2+ and Ca2+ while phenylmethylsulfonyl fluoride (PMSF) inhibited its activity at 0.5 mM. The expressed purified alkaline protease may find potential applications in food and biotechnological industries.

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

&#x0D; 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. &#x0D; Int. J. Appl. Sci. Biotechnol. Vol 8(2): 154-160

Cloning, Expression, and Structural Elucidation of a Biotechnologically Potential Alkaline Serine Protease From a Newly Isolated Haloalkaliphilic Bacillus lehensis JO-26.

An alkaline protease gene of Bacillus lehensis JO-26 from saline desert, Little Rann of Kutch, was cloned and expressed in Escherichia coli BL21 (DE3). A 1,014-bp ORF encoded 337 amino acids. The recombinant protease (APrBL) with Asp 97, His 127, and Ser 280 forming catalytic triad belongs to the subtilase S8 protease family. The gene was optimally expressed in soluble fraction with 0.2 mM isopropyl β-D-thiogalactopyranoside (IPTG), 2% (w/v) NaCl at 28°C. APrBL, a monomer with a molecular mass of 34.6 kDa was active over pH 8–11 and 30°C−70°C, optimally at pH 10 and 50°C. The enzyme was highly thermostable and retained 73% of the residual activity at 80°C up to 3 h. It was significantly stimulated by sodium dodecyl sulfate (SDS), Ca2+, chloroform, toluene, n-butanol, and benzene while completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and Hg2+. The serine nature of the protease was confirmed by its strong inhibition by PMSF. The APrBL gene was phylogenetically close to alkaline elastase YaB (P20724) and was distinct from the well-known commercial proteases subtilisin Carlsberg (CAB56500) and subtilisin BPN′ (P00782). The structural elucidation revealed 31.75% α-helices, 22.55% β-strands, and 45.70% coils. Although high glycine and fewer proline residues are a characteristic feature of the cold-adapted enzymes, the similar observation in thermally active APrBL suggests that this feature cannot be solely responsible for thermo/cold adaptation. The APrBL protease was highly effective as a detergent additive and in whey protein hydrolysis.

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.

Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709

BackgroundBacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis.ResultBacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis.ConclusionWe successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.

Enhancing Activities of Salt-Tolerant Proteases Secreted by Aspergillus oryzae Using Atmospheric and Room-Temperature Plasma Mutagenesis.

Aspergillus oryzae 3.042 was mutagenized using atmospheric and room-temperature plasma (ARTP) technology to enhance its salt-tolerant proteases activity. Compared to the starting strain, mutant H8 subjected to 180 s of ARTP treatment exhibited excellent genetic stability (15 generations), growth rate, and significantly increased activities of neutral proteases, alkaline proteases, and aspartyl aminopeptidase during fermentation. Mutant H8 significantly enhanced the contents of 1-5 kDa peptides, aspartic acid, serine, threonine, and cysteine in soy sauce by 16.61, 7.69, 17.30, 8.61, and 45.00%, respectively, but it had no effects on the contents of the other 14 free amino acids (FAAs) due to its slightly enhanced acidic proteases activity. Analyses of transcriptional expressions of salt-tolerant alkaline protease gene (AP, gi: 217809) and aspartyl aminopeptidase gene (AAP, gi: 6165646) indicated that their expression levels were increased by approximately 30 and 27%, respectively. But no mutation was found in the sequences of AP and AAP expression cassettes, suggesting that the increased activities of proteases in mutant H8 should be partially attributed to the increased expression of proteases. ARTP technology showed great potential in enhancing the activities of salt-tolerant proteases from A. oryzae.

Characterization of a novel bidirectional promoter in Bacillus subtilis

Based on the transcriptome analysis data of a Bacillus licheniformis, a novel bidirectional promoter was identified from the strain and its transcriptional strength was analyzed. The expression level of a Bacillus clausii derived alkaline protease gene driven by the bidirectional promoter was studied by using the known strong constitutive promoter pShuttle-09 as a control. Three recombinant expression vectors and the corresponding recombinant bacteria were constructed. Under the control of the new promoter pLA and its reverse promoter pLB, the alkaline protease expression level respectively reached 164 U/mL and 111 U/mL. The results indicated that the transcription strength of pLA was significantly higher than that of pShuttle-09 and pLB, and both the pLA and pLB promoters could initiate the expression of the alkaline protease. Thus, it provides a new expression element for the heterogenous genes in Bacillus sp. and a new idea for the co-expression of two genes in one prokaryotic strain.

Cloning, expression, purification and characterisation of serine alkaline protease from Bacillus subtilis RD7

Abstract Proteases (E.C 3.4) are enzymes that break peptide bonds between amino acid groups of proteins. Bacillus subtilis RD7, an extracellular protease producer was genetically characterised, B. subtilis genomic DNA was isolated, oligonucleotide primers specific to serine alkaline protease gene of B. subtilis were designed and its PCR amplification was done. The purified PCR product and pET15b vector were subjected to restriction digestion with Nde1 and BamH1 and transformed into Escherichia coli DH5α competent cells. The recombinant expression of serine alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the serine alkaline protease protein had an estimated molecular size of 43 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1203bp gene encoding a protein of 400 amino acids. The sequence was blasted and aligned with known serine alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with closely related subsp. of B. subtilis. The insert also showed many substitutions (mutations with other sp. of Bacillus which established that serine alkaline protease of B. subtilis RD7 is a novel gene. The phylogenetic analysis of serine alkaline protease gene and its predicted amino acid sequences also validated that serine alkaline protease gene is a novel gene and has been accessioned in GenBank with accession number (MN097797). When expressed in E. coli, the recombinant enzyme was over expressed in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by PMSF. The enzyme showed maximum activity at pH 10 and 40 °C. It was stable at pH from 6 to 11 and below 70 °C. The aim of this study is to clone serine alkaline protease gene from B. subtilis RD7 (MG255317), its expression in mesophilic E. coli strain BL21, Purification and characterisation of the expressed protein.

Functional Secretion of Granulocyte Colony Stimulating Factor in Bacillus subtilis and Its Thermogenic Activity in Brown Adipocytes

Granulocyte colony-stimulating factor (G-CSF) is involved in generation of colonies hematopoietic cells by stimulation of bone marrow cells, resulting in the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. To express and secrete G-CSF in Bacillus system, the G-CSF gene was amplified using gene-specific primers and template DNA (pUNO1-hGCSFb). As a result, the PCR product of 530 bp was obtained, sequenced, and analyzed using the BLAST program. The open reading frame encoded a 204-amino acid polypeptide precursor consisting of the 30-residue signal sequence and a 174-residue mature polypeptide. Recombinant secretion vector (pRBAS-CSF3, 6.96 kb) was constructed using pRBAS vector which contains promoter (0.45 kb) and signal sequence (87 bp) of alkaline protease gene (aprE), ribosome binding site (GGAGAGGG) and transferred to Bacillus subtilis LKS. G-CSF protein was secreted and optimized to increase the productivity (19% of total secreted proteins, 0.12 mg/ml). The culture broth was harvested and followed by metal chelating, ethanol precipitation, and gel chromatography. The purified G-CSF was tested for thermogenic activity in cultured brown adipocytes. G-CSF activated HIB1B brown adipocytes by increasing expression levels of brown fat-specific marker proteins (UCP1 and PRDM16) and genes (Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. This result suggests that G-CSF per se or G-CSF-containing foodstuff can be used to treat obesity.

Cloning, expression and characterization of a novel alkaline serine protease gene from native Iranian Bacillus sp.; a producer of protease for use in livestock

The use of proteases in the last decade has been welcomed in livestock and poultry industries and has led to significant results such as improved feed conversion ratio, weight gain and increased growth performance. In the present study, isolation and identification of a novel alkaline protease from Iranian Bacillus species was performed in order to use in livestock feed. After primary isolation of bacteria from soil samples of rice fields and early detection of bacterial genus, the zymogram plate was performed for evaluation of production extracellular proteases. Of the 11 strains producing protease, one strain that produced more enzymes was selected to continue the work. Characterization of alkaline protease was done using specific enzyme assays. To confirm the genus of isolates as well as to identify the species close to, molecular analysis of 16S rRNA gene sequence was done. After that, bioinformatics analysis carried out in NCBI database for searching bacterial alkaline proteases gene sequences. The primer designed based on gene homology of close species for extraction of alkaline protease gene. The results showed that the enzyme extract had the highest activity at pH 9.0 and 50 °C. The 16S rRNA gene sequence was submitted for the strain called Bacillus sp. RAM on the NCBI site. According to the results of the phylogenetic tree, the bacterium was belonged to Bacillus genus and Bacillus sp. RAM was close to Thuringiensis C405. The isolated alkaline protease gene successfully cloned in pET28a and transferred to the expression host E.coli BL21. The expression of the protease gene was evaluated by SDS-PAGE electrophoresis. The induced recombinant cells expressed the protease and revealed molecular weight band of about 38 kDa. According to the enzyme properties, this alkaline protease can useful for application in animal industry.

Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system.

Bacillus amyloliquefaciens K11 is a hyperproducer of extracellular neutral protease, which can produce recombinant homologous protein steadily and is amenable to scale up to high-cell density fermentation. The present study aims to genetically modify strain K11 as a highly efficient secretory expression system for high-level production of heterologous proteins. Using B. amyloliquefaciens K11 and alkaline protease gene BcaprE as the expression host and model gene, the gene expression levels mediated by combinations of promoters PamyQ, PaprE and Pnpr and signal peptides SPamyQ, SPaprE and SPnpr were assessed on shake flask level. The PamyQ-SPaprE was found to be the best secretory expression cassette, giving the highest enzyme activities of extracellular BcaprE (13,800 ± 308U/mL). Using the same expression system, the maltogenic α-amylase Gs-MAase and neutral protease BaNPR were successfully produced with the enzyme activities of 19. ± 0.2U/mL and 17,495 ± 417U/mL, respectively. After knocking out the endogenous neutral protease-encoding gene Banpr, the enzyme activities of BcaprE and Gs-MAase were further improved by 25.4% and 19.4%, respectively. Moreover, the enzyme activities of BcaprE were further improved to 30,200 ± 312U/mL in a 15L fermenter following optimization of the fermentation conditions. In the present study, the genetically engineered B. amyloliquefaciens strain 7-6 containing PamyQ-SPaprE as the secretory expression cassette was developed. This efficient expression system shows general applicability and represents an excellent industrial strain for the production of heterologous proteins.