Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

Purification and Characterization of an Extracellular Alkaline Serine Protease with Dehairing Function from <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography, ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal activity at pH 10 and with a temperature of 55°C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology with protease from another B. pumilus strain. Received: 17 April 2002 / Accepted: 24 May 2002     

Multiple active forms of a novel serine protease from Bacillus subtilis

Summary We have cloned and sequenced a gene (epr) encoding a novel serine protease from Bacillus subtilis. Several active forms of the enzyme with molecular masses between 40 and 34 kDa were found in the medium of B. subtilis cultures containing the epr gene cloned on a plasmid. Deletions at the 3 end of the gene, removing up to 240 amino acids of the reading frame, abolished the expression of the larger species but did not affect the expression of the 34 kDa enzyme. The C-terminal third of the protein is therefore not required for protease activity. The size variation of the active forms expressed by the complete epr gene appears to be the result of partial removal of the C-terminus either by processing or degradation. Thus, the epr gene consists of two domains, one encoding a serine protease homologous to subtilisin and the other a C-terminus of unknown function.Parts of this work were presented at the Fourth International Conference on Genetics and Biotechnology of Bacilli, San Diego, 1987     

Molecular cloning,nucleotide sequence and expression of the structural gene for a thermostable alkaline protease from Bacillus sp. no. AH-101

Summary Alkaliphilic Bacillus sp. no. AH-101 produces an extremely thermostable alkaline serine protease that has a high optimum pH (pH 12–13) and shows keratinolytic activity. The gene encoding this protease was cloned in Escherichia coli and expressed in B. subtilis. The cloned protease was identical to the AH-101 protease in its optimum pH and thermostability at high alkaline pH. An open reading frame of 1083 bases, identified as the protease gene, was preceded by a putative Shine-Dalgarno sequence (AAAGGAGG) with a spacing of 11 bases. The deduced amino acid sequence revealed a pre-pro-peptide of 93 residues followed by the mature protease comprising 268 residues. AH-101 protease showed slightly higher homology to alkaline proteases from alkaliphilic bacilli (61.2% and 65.3%) than to those from neutrophilic bacilli (54.9–56.7%). Also AH-101 protease and other proteases from alkaliphilic bacilli shared common amino acid changes and a four amino acid deletion when compared to the proteases from neutrophilic bacilli. AH-101 protease, however, was distinct among the proteases from alkaliphilic bacilli in showing the lowest homology to the others.Correspondence to: H. Takami     

Overexpression,purification and characterization of a recombinant secretary catalase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

A recombinant Bacillus subtilis strain (KN25) was generated for the large-scale preparation of catalase. The B. subtilis katA gene encoding for catalase was cloned into the shuttle vector PRB374, downstream of the constitutively active vegII promoter, followed by transformation of the B. subtilis strain WB600 with the plasmid. The transformant strain, KN25 secretes high levels (3,500 U/ml) of catalase, which facilitates its purification. Three simple purification steps yielded nearly homogeneous catalase, with ∼70% recovery. The purified recombinant catalase has a specific activity of 34,600 U/mg under optimal conditions, and is more resistant to acidic conditions than bovine liver catalase.     

Enhancement of alkaline protease production in Bacillus circulans using plasmid transformation

Plasmid transformation is an efficient and crucial biotechnological tool that enables the enhancement of many important microbial characters that would be beneficial in a lot of industrial, agricultural and environmental applications. In the present study, five Bacillus species (B. subtilis, B. cereus, B. alvei, B. circulans and B. pumilus) were investigated. They were isolated from agricultural soils of different local arid environments of the Kingdom of Saudi Arabia, identified and characterized for their plasmid content. The main objective of the present study was to enhance the production of alkaline protease in Bacillus circulans (the recipient strain) through plasmid transformation from B. subtilis (the donor strain). All the tested Bacillus strains successfully produced unique multiple (3, 4 and 5) spontaneous antibiotic resistant mutants against chloramphenicol, neomycin, rifampicin, streptomycin, kanamycin and tetracycline and all of which were mutated to Rif_r strains. B. pumilus showed the highest resistance against five of the six tested antibiotics while both of B. alvei and B. circulans showed the lowest resistance to only three of the tested antibiotics. Results revealed that B. subtilis was the best among the tested species concerning the production of alkaline protease (90.2 U/ml) while B. pumilus was the lowest in activity (40.3 U/ml). Screening of plasmid content revealed the presence of one or two mega indigenous plasmids in all the tested species. The four transformant strains BC ₁ , BC ₂ , BC ₃ and BC ₄ resulting from plasmid transformation exhibited significant increases in the activity of alkaline protease and recorded 2.31- to 3-fold increases compared to the parent B. circulans cells and 2.11- to 2.75-fold increases compared to the donor cells of B. subtilis. They also acquired antibiotic resistance to tetracycline and chloramphenicol that was completely absent in the parent cells of B. circulans. Results revealed that plasmid transformation among the tested Bacillus spp. is a powerful technique that can be efficiently exploited to enhance alkaline protease production in the transformed Bacillus spp. compared to their wild strains and we recommend using the improved transformant strains for commercial and industrial purposes.     

Expression of the colH Gene Encoding Clostridium histolyticum Collagenase in Bacillus subtilis and Its Application to Enzyme Purification

The colH gene encoding 116-kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli-Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colH gene was introduced into B. subtilis DB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The yield of rColH from an 800-ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type-I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N-terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N-terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colH gene. Therefore, the recombinant B. subtilis culture is capable of serving as a useful source for enzyme purification.     

Expression system for recombinant human growth hormone production from Bacillus subtilis

We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid‐gene of two DNA fragments, i.e., signal (pre‐) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg‐promoter in B. subtilis. Recombinant‐hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N‐terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal‐peptidase. The highest rhGH concentration was obtained at t = 32 h as C_rhGH = 70 mg L^?1 with a product yield on substrate Y_rhGH/S = 9 g kg^?1, in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic‐acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino‐acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cloning,Characterization, and Expression of the Gene Encoding Alkaline Protease in the Marine Yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> 10

The alkaline protease structural gene (ALP1 gene) was isolated from both the genomic DNA and cDNA of Aureobasidium pullulans 10 by inverse PCR and RT-PCR. An open reading frame of 1248 bp encoding a 415 amino-acid protein with calculated molecular weight of 42.9 kDa was characterized. The gene contained two introns, which had 54 bp and 50 bp, respectively. The promoter of ALP1 gene was located from -62 to -112 and had two CCAAT boxes and one TATA box. The terminator of ALP1gene contained the sequence with a hairpin structure (AAAAAGTT TGGTTTTT). The protein sequence deduced from ALP1 gene exhibited 55.24%, 50.35%, and 31.68% identity with alkaline proteases from Aspergillus fumigatus, Acremonium chrysogenum, and Yarrowia lipolytica, respectively. The protein was found to have the conserved serine active site and histidine active site of serine proteases in the subtilisin family. The recombinant A. pullulans alkaline protease produced in Y. lipolytica formed clear zones on the double plates with 2% casein and alkaline protease activity in the supernatant of the recombinant Y. lipolytica culture was detected, suggesting that the cloned ALP1 gene is expressed in Y. lipolytica and the expressed alkaline protease is secreted into the medium.     

Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

The cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17 was cloned on separatePstI,BamHI, andEcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host,Escherichia coli DH5. High level constitutive expression of the gene product was also detrimental to theE. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kbEcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the hostB. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in theB. subtilis host; however, expression was at a low level. Subcloning of the 3-kbEcoRI fragment into pUC18 and transformation intoE. coli XL1-Blue (FlacI^q) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from theBacillus temperate phage SPO2 promoter of pPL708 may increase expression of this gene.Florida Agricultural Experiment Station Journal Series No. R-02177     

Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906

Bacillus circulans MTCC 7906, an extracellular alkaline protease producer was genetically characterized. B. circulans genomic DNA was isolated, oligonucleotide primers specific to alkaline protease gene of B. circulans were designed and its PCR amplification was done. The purified PCR product and pTrcHisA vector were subjected to restriction digestion with NcoI and HindIII and transformed into Escherichia coli DH5-α competent cells. The recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the alkaline protease protein had an estimated molecular size of 46 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1329 bp gene encoding a protein of 442 amino acids. The sequence was blasted and aligned with known alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with three closely related subsp. of B. subtilis (B. subtilis subsp. subtilis strain 168, B. subtilis BSn5 and B. subtilis subsp. spizizenii strain W23). The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The phylogenetic analysis of alkaline protease gene and its predicted amino acid sequences also validated that alkaline protease gene is a novel gene and the same has been accessioned in GenBank with accession number {"type":"entrez-nucleotide","attrs":{"text":"JN645176.1","term_id":"353260575","term_text":"JN645176.1"}}JN645176.1.     

Cloning of <Emphasis Type="Italic">srfA</Emphasis> operon from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C9 and its expression in <Emphasis Type="Italic">E. coli</Emphasis>

The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound extracted from the E. coli transformant exhibited a different R _f value of 0.52 from B. subtilis C9 or authentic surfactin (R _f = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon.     

Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris

The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644?U?mL^?1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2?kDa and 29.6?kDa, both larger than the predicted mass of 22.4?kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6–9 and 50°C, respectively.     

Gene Cloning,Expression, and Properties of a Fibrinolytic Enzyme Secreted by <Emphasis Type="Italic">Bacillus pumilus</Emphasis> BS15 Isolated from Gul (Oyster) Jeotgal

A Bacillus strain, BS15, showing strong fibrinolytic activity, antibacterial activity, and salt tolerance was isolated from gul (oyster) jeotgal, a Korean fermented sea food. BS15 was identified as B. pumilus. B. pumilus BS15 was able to grow in LB broth with 18% (w/v) NaCl. When culture supernatant was analyzed by SDS-PAGE, 22, 27, 35, and 60 kDa proteins were observed. The 27 kDa protein was determined to be major fibrinolytic enzyme by fibrin zymography. The gene (aprEBS15) was cloned in pHY300PLK, a Bacillus-E. coli shuttle vector. A B. subtilis transformant (TF) harboring pHYBS15 showed higher fibrinolytic activity than B. pumilus BS15, and produced the same 27 kDa protein. aprEBS15 was overexpressed in E. coli BL21 (DE3), and recombinant enzyme (AprEBS15) was purified. The optimum pH and temperature of AprEBS15 were pH 8.0 and 40°C, respectively. Km and Vmax values were 0.26 mM and 21.88 µmol/L/min, respectively. B. pumilus BS15 can be used as a starter for jeotgals and other fermented foods with high salinities.     

Cloning of the gene responsible for the extracellular proteolytic activities of Bacillus licheniformis

Summary Effect of the cloned gene of Bacillus licheniformis on the extracellular proteolytic activities of B. subtilis was investigated. The gene was cloned onto the vector plasmid pUB110 (3.0 Md), and the introduction of the hybrid plasmid [pAN2 (5.4 Md)] into the cells of B. subtilis resulted in a marked increase of activities of the extracellular alkaline and neutral proteases, which had optimal pHs at 10.5 and 7.2, respectively. On DEAE-Sephadex column chromatography, the extracellular activity of B. subtilis with pAN2 was separated into two active fractions (a₁ and b₁). The activity in a₁ was specifically inactivated by diisopropyl phosphorofluoridate (DFP) and tosyl fluoride (TSF), potent inhibitors of alkaline proteases, while, the activitiy in b₁ was inhibited by ethylenediaminetetraacetate (EDTA), an inhibitor of neutral protease, but not by DEP or TSF.Sub-cloning with genes shortened to about 0.85 Md (pAN2-1) and 0.25 Md (pAN2-2) increased the activities of both alkaline and neutral proteases. The extracellular -amylase and ribonuclease production was also increased when the host strain was transformed with these hybrid plasmids (pAN2, pAN2-1, pAN2-2). The increase in activity of proteases by the cloning was discussed in relation to regulation of the production and/or secretion of the enzyme.     

n-Terminal Amino Acid Sequences of Neutral Proteases from Bacillus amyloliquefaciens and Bacillus subtilis: Identification of a Neutral Protease Gene Cloned in Bacillus subtilis

Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures.

The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini.

Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.     

Influence of a Cell-Wall-Associated Protease on Production of α-Amylase by Bacillus subtilis

AmyL, an extracellular α-amylase from Bacillus licheniformis, is resistant to extracellular proteases secreted by Bacillus subtilis during growth. Nevertheless, when AmyL is produced and secreted by B. subtilis, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the B. subtilis wprA gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible wprA gene, we show that a product of wprA, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in which wprA is not expressed exhibits an increased yield of α-amylase. The potential role of wprA in protein secretion is discussed, together with implications for the use of B. subtilis and related bacteria as hosts for the secretion of heterologous proteins.     

Cloning and overexpression of <Emphasis Type="Italic">aprE3-17</Emphasis> encoding the major fibrinolytic protease of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> CH 3-17

Bacillus licheniformis (B. licheniformis) CH3-17, an isolate from cheonggukjang, a traditional Korean fermented soyfood, secretes several fibrinolytic enzymes into the culture medium, showing strong fibrinolytic activity. A gene homologous to aprE of Bacillus subtilis (B. subtilis), aprE3-17, was cloned by PCR. DNA sequencing showed that aprE3-17 encodes a prepro-type serine protease consisting of 382 amino acids. The mature enzyme was 27 kDa in size. The aprE3-17 gene was overexpressed in B. subtilis WB600 using pHY300PLK, an Escherichia coli (E. coli)-Bacillus shuttle vector, and the 27 kDa enzyme was purified from the culture supernatant. The optimum pH for activity was 6.0. Purified enzyme quickly degraded the Aα and Bβ chains of fibrinogen but could not degrade the γ-chain.     

A new organic solvent tolerant protease from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> 115b

Five out of the nine benzene–toulene–ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.     

Efficient expression of an alkaline pectate lyase gene from Bacillus subtilis and the characterization of the recombinant protein

The gene encoding a novel alkaline pectate lyase (Apel) from Bacillus subtilis was cloned and expressed in B. subtilis WB600. Apel contained an ORF of 1,260 bp, encoding a signal peptide of 21 amino acids and a mature protein of 399 amino acids with a calculated molecular mass of 45497.9 Da. The mature Apel was structurally related to the enzymes in the polysaccharide lyase family 1. After purification, the recombinant Apel had a specific activity of 445 U mg⁻¹. The enzyme was optimally active at 50°C and pH 9.