Extracellular Neutral Protease Research Articles

Production of Protease on Wheat Bran by a Newly Isolated Streptomyces SP. Under Solid State Fermentation

Proteases are a group of enzymes that catalyse the degradation of proteins resulting in the production of their amino acid constituents. In the present study newly isolated Streptomyces sp. was subjected to produce proteases through solid state fermentation while wheat bran was used as substrate. To produce proteases, a local strain Streptomyces sp. was isolated from a soil sample of Ezzemouls saltpan, located in Ain M'lila (East of Algeria). The phenotypic and phylogenetic studies of this strain showed that it represents probably a new species. The SSF production medium for Streptomyces sp. was optimized using Plackett and Burman statistical methods. The results showed a maximum activity on basal wheat bran medium supplemented with 1% fructose. The best SSF humidifying solution was pH 9.0 phosphate buffer at 50% moisture. Protease has an optimum at pH 7.0, which is a typical characteristic of neutral proteases. The optimum temperature was 60°C and proved stable up to 80°C. The results showed that the novel Streptomyces sp. isolate is a good producer of extracellular neutral protease on wheat bran, which can be beneficial for industries. J. Bio-Sci. 29(1): 33-48, 2021 (June)

Production of an extracellular neutral protease by Bacillus aerius UB02 endophytic to carnivorous plant Utricularia stellaris

Aim: Endophytic bacteria indigenous to carnivorous plants have been explored for production of novel bioactive metabolites including extracellular enzymes. Bacillus aerius UB02, an extracellular neutral protease producing isolate endophytic to bladder of Utricularia stellaris L. f. was used in this study. Methodology: The bacterial isolate UB02 was identified following morpholological, physiological, biochemical and 16S rRNA gene sequence analyses. The media as well as the cultural conditions for production of protease were optimized. The extracellular protease was isolated and purified from the cell-free culture filtrate by ammonium sulphate precipitation, dialysis and DEAE Sephadex ion exchange column chromatography and the optimum conditions for its activity were determined. Results: The isolate Bacillus aerius UB02 (GenBank accession no. MK 696417, MCC accession no. 4132), produced significant amount of extracellular protease (38.29 U mg-1 protein) during growth in casein supplemented synthetic medium. However, peptone yeast extract glucose medium appeared to be the best for the synthesis of enzyme. Production of enzyme was enhanced by the inoculum density of 1.5% (v/v), culture volume: flask volume (CVF) ratio of 1:10, substrate concentration of 2.5% (w/v) with temperature and pH adjusted at 37°C and 7.4, respectively. Glucose (2.2%, w/v) and ammonium chloride (1.2 g/L) as carbon and nitrogen sources also favoured the enzyme production. The neutral protease with a molecular weight of approximately 35 kDa showed maximum activity at 40°C, pH 7.8 with 2% (w/v) casein. The enzyme exhibited Km and Vmax values of 6.81 mg ml-1 and 62.5 U mg-1 of protein, respectively, and was moderately thermostable. The protease activity was inhibited by Pb and Cd as well as 1,10-phenanthroline and β-mercaptoethanol. Interpretation: These findings will help not only in understanding the role of endophytic bacteria and the enzymes produced by them in the digestion of prey by carnivorous plant but could also be explored for application in the field of biotechnology.

Integrated Transcriptomic and Proteomic Analyses Reveal the Role of NprR in Bacillus anthracis Extracellular Protease Expression Regulation and Oxidative Stress Responses.

NprR is a protein of Bacillus anthracis that exhibits moonlighting functions as either a phosphatase or a neutral protease regulator that belongs to the RNPP family. We previously observed that the extracellular protease activity of an nprR deletion mutant significantly decreased within in vitro cultures. To identify the genes within the regulatory network of nprR that contribute to its protease activity, integrated transcriptomic and proteomic analyses were conducted here by comparing the nprR deletion mutant and parent strains. A total of 366 differentially expressed genes (DEGs) between the strains were observed via RNA-seq analysis. In addition, label-free LC-MS/MS analysis revealed 503 differentially expressed proteins (DEPs) within the intracellular protein fraction and 213 extracellular DEPs with significant expressional differences between the strains. The majority of DEGs and DEPs were involved in environmental information processing and metabolism. Integrated transcriptomic and proteomic analyses indicated that oxidation-reduction-related GO terms for intracellular DEPs and endopeptidase-related GO terms for extracellular DEPs were significantly enriched in the mutant strain. Notably, many genes involved in protease activity were largely downregulated in the nprR deletion mutant cultures. Moreover, western blot analysis revealed that the major extracellular neutral protease Npr599 was barely expressed in the nprR deletion mutant strain. The mutant also exhibited impaired degradation of protective antigen, which is a major B. anthracis toxin component, thereby resulting in higher protein yields. Concomitantly, another global transcriptional regulator, SpxA1, was also dramatically downregulated in the nprR deletion mutant, resulting in higher sensitivity to oxidative and disulfide stress. These data consequently indicate that NprR is a transcriptional regulator that controls genes whose products function as extracellular proteases and also is involved in oxidative stress responses. This study thus contributes to a more comprehensive understanding of the biological function of NprR, and especially in the middle growth stages of B. anthracis.

Extracellular neutral protease from Arthrospira platensis: Production, optimization and partial characterization

Proteases are industrially important catalysts. They belong to a complex family of enzymes that perform highly focused proteolysis functions. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. In the present study, a novel extracellular neutral protease produced from Arthrospira platensis was detected and characterized. Its proteolytic activity was strongly activated by β-mercaptoethanol, 5,5-dithio-bis-(2-nitrobenzoic acid) and highly inhibited by Hg2+ and Zn2+ metal ions which support the fact that the studied protease belongs to the cysteine protease family. Using statistical modelling methodology, the logistic model has been selected to predict A. platensis growth-kinetic values. The optimal culture conditions for neutral protease production were found using Box-Behnken Design. The maximum experimental protease activities (159.79 U/mL) was achieved after 13 days of culture in an optimized Zarrouk medium containing 0.625 g/L NaCl, 0.625 g/L K2HPO4 and set on 9.5 initial pH.The extracellular protease of A. platensis can easily be used in the food industry for its important activity at neutral pH and its low production cost since it is a valuation of the residual culture medium after biomass recovery.

Engineering a recombination neutral protease I from Aspergillus oryzae to improve enzyme activity at acidic pH.

Extracellular neutral proteases (NPs) in Aspergillus oryzae (A. oryzae) play a role in hydrolyzing soybean proteins into smaller peptides at pH about 7.5. The optimum pH of moromi fermentation (The second stage of soy sauce fermentation.) is 4.5–5.5. NPI is acid sensitive. To decrease the pH optimum of NPI, we got a mutant NPI-Y122FK246ID382V from the error-prone PCR library that showed optimal activity at pH 5.5. The specific activity at 40 °C of the NPI-Y122FK246ID382V mutant was 1383.50 U mg−1, which was 2.75-fold that of wild-type (503.09 U mg−1). The Michaelis constants of the mutant decreased from 22.13 mM (wild-type) to 19.98 mM (NPI-Y122FK246ID382V). The residues at positions 122 and 246 are important in influencing hydrolytic activity at pH 5.5 through site-directed mutagenesis. And the pH optimum of double amino acid mutants (Y122FK246I) shifted dramatically to an acidic pH compared to those of single amino acid substitution. Molecular models and structural comparisons of native and mutant provided further insight on the basis to improve catalytic efficiency at acidic pH. These results indicated that we modified the neutral protease I of Aspergillus oryzae, which can effectively improve the application of the neutral protease in industrial production, and finally lay the foundation for improving the utilization rate of raw protein.

Biofilm-associated toxin and extracellular protease cooperatively suppress competitors in Bacillus subtilis biofilms.

In nature, most bacteria live in biofilms where they compete with their siblings and other species for space and nutrients. Some bacteria produce antibiotics in biofilms; however, since the diffusion of antibiotics is generally hindered in biofilms by extracellular polymeric substances, i.e., the biofilm matrix, their function remains unclear. The Bacillus subtilis yitPOM operon is a paralog of the sdpABC operon, which produces the secreted peptide toxin SDP. Unlike sdpABC, yitPOM is induced in biofilms by the DegS-DegU two-component regulatory system. High yitPOM expression leads to the production of a secreted toxin called YIT. Expression of yitQ, which lies upstream of yitPOM, confers resistance to the YIT toxin, suggesting that YitQ is an anti-toxin protein for the YIT toxin. The alternative sigma factor SigW also contributes to YIT toxin resistance. In a mutant lacking yitQ and sigW, the YIT toxin specifically inhibits biofilm formation, and the extracellular neutral protease NprB is required for this inhibition. The requirement for NprB is eliminated by Δeps and ΔbslA mutations, either of which impairs production of biofilm matrix polymers. Overexpression of biofilm matrix polymers prevents the action of the SDP toxin but not the YIT toxin. These results indicate that, unlike the SDP toxin and many conventional antibiotics, the YIT toxin can pass through layers of biofilm matrix polymers to attack cells within biofilms with assistance from NprB. When the wild-type strain and the YIT-sensitive mutant were grown together on a solid medium, the wild-type strain formed biofilms that excluded the YIT-sensitive mutant. This observation suggests that the YIT toxin protects B. subtilis biofilms against competitors. Several bacteria are known to produce antibiotics in biofilms. We propose that some bacteria including B. subtilis may have evolved specialized antibiotics that can function within biofilms.

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.

Purification and Biochemical Characterization of a Neutral Serine Protease from Trichoderma harzianum. Use in Antibacterial Peptide Production from a Fish By-Product Hydrolysate.

This study reports the purification and biochemical characterization of an extracellular neutral protease from the fungus Trichoderma harzianum. The protease (Th-Protease) was purified from the culture supernatant to homogeneity by a three-step procedure with 14.2% recovery and 9.06-fold increase in specific activity. The purified enzyme appeared as a single protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of about 20kDa. The optimum pH and temperature for the proteolytic activity were pH 7.0 and 40°C, respectively. The enzyme was then investigated for its potential application in the production of antibacterial peptides. Interestingly, Scorpaena notata viscera protein hydrolysate prepared using the purified serine protease (Th-Protease) showed remarkable in vitro antibacterial activities. A peptide with a high antibacterial activity was further purified by a three-step procedure, and its sequence was identified as FPIGMGHGSRPA. The result of this study offers a promising alternative to produce natural antibacterial peptides from fish protein hydrolysate.

Neutral serine protease from Penicillium italicum. Purification, biochemical characterization, and use for antioxidative peptide preparation from Scorpaena notata muscle.

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0-9.0. The protease was activated by divalent cations such as Ca(2+) and Mg(2+). Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.

Chryseobacterium sp. JK1이 분비하는 세포외 단백질분해효소 특성

이전의 연구에서 토양으로부터 많은 양의 세포외 단백질분해 효소를 생산하는 신종 중온세균 Chryseobacterium sp. JK1를 분리하였다. 이 균주가 생산하는 단백질 분해효소의 특성조사 결과 최적반응온도와 pH는 각각 <TEX>$40^{\circ}C$</TEX>와 7.0이였으며, 좁은 최적온도 구간과 비교적 넓은 pH 구간인 pH 6.0-9.0에서 높은 활성을 보여주었다. 그리고 단백질 분해효소는 EDTA 또는 EGTA, PMSF와 금속이온 <TEX>$Ag^+$</TEX> 또는 <TEX>$Cu^{2+}$</TEX>의 첨가에 의해 강하게 저해 되었으며, <TEX>$Al^{3+}$</TEX>의 첨가에 의해 약하게 저해되었다. Pepstatin과 금 속이온 <TEX>$K^+$</TEX>, <TEX>$Ca^{2+}$</TEX>, <TEX>$Na^+$</TEX>, <TEX>$Fe^{2+}$</TEX> 또는 <TEX>$Mg^{2+}$</TEX>의 첨가는 저해에 큰 영향을 주지 않았다. 이와 반대로 단백질분해효소는 이가 금속이온인 <TEX>$Mn^{2+}$</TEX> (5 mM)의 첨가에 의해 효소활성이 향상되었다. 농축된 배양 상등액의 활성염색 분석으로 67과 145 kDa 크기의 주요 밴드 두 개가 관찰되었다. 이러한 결과들로 Chryseobacterium sp. JK1 균주가 식품산업에 응용 가능한 세포외 중성의 serine 단백질 분해효소를 생산한다는 것을 알 수 있었다. A novel Chryseobacterium sp. JK1 strain isolated from soil had been reported that this isolate produced large amount of extracellular protease at mesophilic temperature in previous study. The optimal temperature and pH of extracellular protease were <TEX>$40^{\circ}C$</TEX> and 7.0, respectively, showing narrow range of optimal temperature and relatively broad activity from pH 6.0 to 9.0. In addition, the protease showed greatest activity against skim milk and lowest against bovine serum albumin (BSA). The protease strongly inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) or phenylmethylsulfonyl fluoride (PMSF), and addition of cation <TEX>$Ag^+$</TEX> or <TEX>$Cu^{2+}$</TEX>, and slightly inhibited by <TEX>$Al^{3+}$</TEX>. No significant inhibition was found with pepstatin, and addition of cation, <TEX>$K^+$</TEX>, <TEX>$Ca^{2+}$</TEX>, <TEX>$Na^+$</TEX>, <TEX>$Fe^{2+}$</TEX> or <TEX>$Mg^{2+}$</TEX>. On the contrary, protease was enhanced by addition of divalent cation <TEX>$Mn^{2+}$</TEX> (5 mM). Zymography analysis of concentrated culture supernatant revealed two major bands at 67 and 145 kDa. These results suggest that Chryseobacterium sp. JK1 strain produced extracellular neutral serine proteases which could apply in food industry.

HtrA is a major virulence determinant of Bacillus anthracis

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.

Role of an extracellular neutral protease in infection against nematodes by Brevibacillus laterosporus strain G4

Proteases have been proposed as virulence factors in microbial pathogenicity against nematodes. However, what kinds of extracellular proteases from these pathogens and how they contribute to the pathogenesis of infections against nematode in vivo remain largely unknown. A previous analysis using a strain with a deletion in an extracellular alkaline protease BLG4 gene from Brevibacillus laterosporus demonstrated that BLG4 was responsible for the majority of nematicidal activity by destroying host's cuticle. In recent studies, a neutral protease NPE-4, purified from the mutant BLG4-6, was found to be responsible for the majority of the remaining EDTA-inhibited protease activity. However, the purified NPE-4 and recombinant NPE-4 in a related species Bacillus subtilis showed little nematicidal activity in vitro and were unable to degrade the intact cuticle of the host. It is interesting to note that the addition of NPE-4 improved the pathogenicity of crude enzyme extract from wild-type B. laterosporus but had no effect on the BLG4-deficient mutant. This result suggests that NPE-4 functions in the presence of protease BLG4. Moreover, NPE-4 could degrade proteins from the inner layer of purified cuticles from nematode Panagrellus redivivus in vitro. These results indicated that the two different bacterial extracellular proteases might play differential roles at different stages of infection or a synthetic role in penetration of nematode cuticle in B. laterosporus. This is among the first reports to systematically evaluate and define the roles of different bacterial extracellular proteases in infection against nematodes.

Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674.

Bacillus cereus KCTC 3674 excretes several kinds of extracellular proteases into the growth medium. Two proteases with molecular masses of approximately 36-kDa and 38-kDa, as shown by SDS-PAGE, were purified from the culture broth. The 38-kDa protease was purified from B. cereus cultivated at 37 degrees C, and the 36-kDa protease was obtained from the B. cereus cultivated at 20 degrees C. The 38-kDa protease was identified as an extracellular neutral (metallo-) protease and was further characterized. The 36-kDa protease was shown to be a novel enzyme based on its N-terminal amino acid sequence, its identification as a metallo-enzyme that was strongly inhibited by EDTA and o-phenanthroline, its hemolysis properties, and its optimal pH and temperature for activity of 8.0 and 70 degrees C, respectively.

Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization.

A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of the B. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.

Evaluation of genotoxic, mutagenic and antitumor properties of 2-amino-2-thiazoline, L-thioproline and 2-amino-2-thiazoline ε-formylaminocaproate

In the present study the genotoxic, mutagenic and antitumor effects of 2-amino-2-thiazoline (R), L-thioproline (N) and 2-amino-2-thiazoline e-formylaminocaproate (FAT) as well as their influence on bacterial growth and activity of the extracellular protease are discussed. On the basis of our study the following conclusions may be drawn: the investigated compounds, except N, had no strongly expressed genotoxic activity and none of them was found to be mutagenic. R and FAT were insignificantly toxic on bacterial growth, all compounds were ineffective against transplanted tumors and did not change the rate of the biosynthesis of Bacillus subtilis extracellular neutral protease.

Modular expression and secretion vectors for Bacillus subtilis

A modular vector system has been developed for the extracellular production of heterologous proteins in Bacillus subtilis. This modular vector system consists of four secretion vectors which are based upon the genes encoding the Bacillus amyloliquefaciens extracellular alkaline protease, neutral protease, barnase and levansucrase. The modular vectors contain compatible restriction sites downstream from the signal peptide-coding region. Three reporter proteins (staphylococcal protein A, levansucrase and Escherichia coli alkaline phosphatase) that offer complementary advantages for cloning, genetic manipulations and media optimization have been fused to the various signal peptides. These secretion vectors function in E. coli and hence can be used to compare the mechanisms of protein secretion in E. coli and B. subtilis.

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus

The gene ( nprV), encoding the extracellular neutral protease, vibrilysin (NprV), of the Gram − marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl acryloyl]- l - alanyl- phenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long ‘pro’ sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified fom cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the ‘pro’ and mature enzyme downstream from a Bacillus promoter and signal sequence.

Proteases involved in generation of beta- and alpha-amylases from a large amylase precursor in Bacillus polymyxa

The genes for extracellular neutral protease (Npr) and intracellular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple beta-amylases and an alpha-amylase from a large amylase precursor. The npr gene was composed of 1,770 bp and 570 amino acids, while the isp gene was composed of 978 bp and 326 amino acids. Both proteases produced by E. coli cleaved the amylase precursor to generate beta- and alpha-amylases. Furthermore, several other proteases produced the same products from the precursor. A 130-kDa amylase precursor has two large domain structures responsible for the generation of beta- and alpha-amylases. The junction region of approximately 200 amino acids may be exposed on the surface of the molecule and susceptible to proteolytic enzymes, which results in the formation of multiple amylases.

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis

We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacU(h)h mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65 degrees C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.

Use of a Triple Portease-deficient Mutant of Bacillus subtilis as a Host for Secretion of a B. subtilis Cellulase and TEM .BETA.-Lactamase.

The mature portion of the TEM beta-lactamase (BLA) gene (bla) derived from pBR322 was fused with the promoter and signal region of Bacillus subtilis cellulase (BSC) gene (bsc), and the productivity was compared with that of the cloned native bsc gene, using a wild-type B. subtilis strain and a strain deficient in three proteases (i.e., extracellular serine protease, extracellular neutral protease, and the major intracellular serine protease) as hosts. The effects of the sen, sacQ, and prtR genes, carried on the same plasmids, were tested as for the productivities of BSC and BLA. The production of BSC was increased 9-fold by using the combination of the triple-protease deficient strain and the sacQ gene, compared with that by using the wild-type strain as a host, and no degradation of BSC was observed in the triple-protease deficient strain. On the other hand, though the production of the BSC-BLA fusion protein increased 2.5-fold in the triple-protease deficient strain, the BLA activity was decreased after the cell reached the stationary phase of growth, possibly due to some proteolysis. These observations show different sensitivity of secretary proteins to cellular proteases, and suggest that BLA is decomposed by remaining minor proteases.