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1.
Okuda M Sumitomo N Takimura Y Ogawa A Saeki K Kawai S Kobayashi T Ito S 《Extremophiles : life under extreme conditions》2004,8(3):229-235
Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394–2,424 bp encoded prosubtilisins of 798–808 amino acids (aa) consisting of the prepropeptides of 151–158 aa and the mature enzymes of 640–656 aa. The deduced aa sequences of the mature enzymes exhibited 60–95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5–11.0, optimal temperatures of 40–45°C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.Communicated by K. Horikoshi 相似文献
2.
Disney Ribeiro Dias Danielle Marques Vilela Marialice Pinto Coelho Silvestre Rosane Freitas Schwan 《World journal of microbiology & biotechnology》2008,24(10):2027-2034
Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion
of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with
three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic
activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms,
a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg
for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media
tested with B.
sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained
from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the
industry to utilize cheese whey to produce proteolytic enzymes. 相似文献
3.
《Bioscience, biotechnology, and biochemistry》2013,77(9):1455-1460
The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus suhtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6—61.70/0). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli. 相似文献
4.
Kobayashi T Lu J Li Z Hung VS Kurata A Hatada Y Takai K Ito S Horikoshi K 《Applied microbiology and biotechnology》2007,75(1):71-80
A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic
Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal
caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed
by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed
in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity
and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus
97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled
to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate
homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins
and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported
from a strict anaerobe in this family. 相似文献
5.
Katsuhisa Saeki Marietta V. Magallones Yasushi Takimura Yuji Hatada Tohru Kobayashi Shuji Kawai Susumu Ito 《Current microbiology》2003,47(4):0337-0340
The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.Received: 19 November 2002 / Accepted: 19 December 2002 相似文献
6.
To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession number D37921. 相似文献
7.
Chu WH 《Journal of industrial microbiology & biotechnology》2007,34(3):241-245
Thirty-five strains capable of secreting extracellular alkaline proteases were isolated from the soil and waste water near
the milk processing plant, slaughterhouse. Strain APP1 with the highest-yield alkaline proteases was identified as Bacillus sp. The cultural conditions were optimized for maximum enzyme production. When the initial pH of the medium was 9.0, the
culture maintained maximum proteolytic activity for 2,560 U ml−1 at 50°C for 48 h under the optimized conditions containing (g−1): soyabean meal, 15; wheat flour, 30; K2HPO4, 4; Na2HPO4, 1; MgSO4·7H2O, 0.1; Na2CO3, 6. The alkaline protease showed extreme stability toward SDS and oxidizing agents, which retained its activity above 73
and 110% on treatment for 72 h with 5% SDS and 5% H2O2, respectively. 相似文献
8.
Tian B Yang J Lian L Wang C Li N Zhang KQ 《Applied microbiology and biotechnology》2007,74(2):372-380
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. 相似文献
9.
Hideto Takami Tetsuo Kobayashi Rikizo Aono Koki Horikoshi 《Applied microbiology and biotechnology》1992,38(1):101-108
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 相似文献
10.
A general activity probe was synthesized and applied to the supernatant of a filamentous fungus, Ophiostoma, culture to identify directly the secreted serine proteases by covalent enzyme labeling. The activity probe contained a chemically
reactive group that reacted with, and thus covalently labeled, the serine residues of only active proteases and not heat-inactivated
proteases. The activity probe also contained a fluorescent group that allowed for the subsequent visualization of the captured
proteases in 1-D gels and their identification by N-terminal sequencing. This use of a chemical probe led to the rapid discovery of subtilisin-like serine protease of Ophiostoma. Two hypothetical proteins were also captured, with one being a probable endopeptidase K type of protease. 相似文献
11.
Chen XG Stabnikova O Tay JH Wang JY Tay ST 《Extremophiles : life under extreme conditions》2004,8(6):489-498
A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH4Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h. 相似文献
12.
Wei Zhang Cao Yueqing Xia Yuxian 《World journal of microbiology & biotechnology》2008,24(11):2481-2488
The subtilisin-like protease Pr1A plays a role in insect cuticle breach and has been used in the development of advanced engineered
biopesticides. We have identified and cloned the Pr1A gene from a locust specific Metarhizium anisopliae strain, CQMa102. The cDNA of Pr1A and its deduced protein sequence were deposited in GenBank (accession numbers EF627449
and ABR20899, respectively). Sequence analysis reveals that Pr1A belongs to the subtilisin-like serine protease family. Analysis
of homologous species shows that the protein exhibits 99% identity with the subtilisin Pr1A from M. anisopliae var. acridum strain FI-985. The CQMa102 Pr1A protein was expressed in Pichia pastoris to verify its protease activity. Our results show that the Pr1A gene cloned from M. anisopliae strain CQMa102 has cuticle-degrading function and is a potential virulence factor for the development of engineered biopesticides. 相似文献
13.
Kamal Kumar B Balakrishnan H Rele MV 《Journal of industrial microbiology & biotechnology》2004,31(2):83-87
Alkaline xylanases from alkaliphilic
Bacillus strains NCL (87-6-10) and Sam III were compared with the commercial xylanases Pulpzyme HC and Biopulp for their compatibility with detergents and proteases for laundry applications. Among the four xylanases evaluated, the enzyme from the alkaliphilic
Bacillus strain NCL (87-6-10) was the most compatible. The enzyme retained its full activity (40 °C for 1 h) in the presence of detergents, whereas Pulpzyme HC and Sam III showed only 30% and 50% of their initial activity, respectively. Biopulp, though stable to detergents, had only marginal activity (5%)at pH 10. However, all four enzymes retained significant activity (80%) for 60 min in the presence of the proteases Alcalase and Conidiobolus protease. Supplementation of the enzyme enhanced the cleaning ability of the detergents. 相似文献
14.
Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH4)2SO4 and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was
observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The
enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate
and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases
from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus. 相似文献
15.
Insect midgut proteases are excellent targets for insecticidal agents such as Bacillus
thuringiensis Cry toxins and protease inhibitors. The midgut proteases of Achaea janata have been characterized and Casein zymograms indicated at least five distinct activities corresponding to approx 17, 20,
29 and 80, and 90 kDa. Using a combination of synthetic substrates and specific inhibitors in casein zymograms, photometric
assays and activity blots, three trypsin-like and one elastase-like serine proteases were identified but no chymotrypsin-like
activity. Various proteinase inhibitors displayed differential inhibitory effects towards the midgut proteases. 相似文献
16.
Qiuhong N Xiaowei H Baoyu T Jinkui Y Jiang L Lin Z Keqin Z 《Applied microbiology and biotechnology》2006,69(6):722-730
An endospore-forming bacterium, strain B16, was isolated from a soil sample and identified as a Bacillus sp. The strain presented remarkable nematotoxic activity against nematode Panagrellus redivivus. The crude extracellular protein extract from culture supernatant of the bacteria killed about 80% of the tested nematodes
within 24 h, suggesting the involvement of extracellular proteases. A homogeneous extracellular protease was purified by chromatography,
and the hypothesis of proteinaceous pathogeny in the infection of B16 strain was confirmed by the experiments of killing living
nematodes and by the degradation of purified nematode cuticle when treated with the homogenous protease. The gene for the
virulence protease was cloned, and the nucleotide sequence was determined. The deduced amino acid sequence showed significant
similarity with subtilisin BPN' but low homology with the other cuticle-degrading proteases previously reported in fungi.
Characterization of the purified protease revealed the molecular mass of 28 kDa and the optimum activity at pH 10, 50°C. The
purified protease can hydrolyze several native proteinaceous substrates, including collagen and nematode cuticle. To our knowledge,
this is the first report of a serine protease from a Bacillus genus of bacteria that serves as a pathogenic factor against nematodes, an important step in understanding the relationship
between bacterial pathogen and host and in improving the nematocidal activity in biological control.
Niu Qiuhong and Huang Xiaowei contributed equally to the work 相似文献
17.
Keiji Nakamura Ayako Matsushima Koki Horikoshi 《Bioscience, biotechnology, and biochemistry》2013,77(6):1261-1267
The state of amino acid residues in alkaline protease of Bacillus No. 221 and that of subtiiisin BPN’ were compared by spectrophotometric tiiration of tyrosine residues and by several reagents: β-naphtoqumone-4,6-disulfonic acid and monochlorofluoroquinone for amino groups, H2O2-dioxane for tryptophan, glyoxal for arginine, and tetranitromethane for tyrosine.The reactivity of both proteases was fairly similar to those reagents.The helix content of alkaline protease of Bacillus No. 221 (37%) was higher than that of subtilisin BPN’ (20%).The Km and Vmax of alkaline protease of Bacillus No. 221 toward ATEE and BTEE were obtained from Lineweaver-Burk plot and compared with those of α-chymotrypsin and subtiiisin BPN’. 相似文献
18.
Juan Li Li Yu Jinkui Yang Linqian Dong Baoyu Tian Zefen Yu Lianming Liang Ying Zhang Xu Wang Keqin Zhang 《BMC evolutionary biology》2010,10(1):68
Background
Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi. 相似文献19.
C. R. Tremacoldi R. Monti H. S. Selistre-De-Araújo E. C. Carmona 《World journal of microbiology & biotechnology》2007,23(2):295-299
An extracellular alkaline serine protease has been purified from a strain of Aspergillus clavatus, to apparent homogeneity, by ammonium sulfate precipitation and chromatography on Sephadex G-75. Its molar mass, estimated
by SDS-PAGE, was 35 kDa. Maximum protease activity was observed at pH 9.5 and 40°C. The enzyme was active between pH 6.0 and
11.0 and was found to be unstable up to 50°C. Calcium at 5 mM increased its thermal stability. The protease was strongly inhibited
by PMSF and chymostatin as well as by SDS, Tween 80 and carbonate ion. Substrate specificity was observed with N-p-Tos-Gly-Pro-Arg-p-nitroanilide and N-Suc-Ala-Ala-Ala-p-nitroanilide being active substates. Parts of the amino acid sequence were up to 81% homologous with those of several fungal
alkaline serine proteases. 相似文献
20.
Yun Sook Kim Dae-Sung Lee Seong-Yun Jeong Woe Jae Lee Myung-Suk Lee 《Journal of microbiology (Seoul, Korea)》2009,47(1):9-18
A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of ULjin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the
lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal
activity by different culture filtrate volumes, the 10% (100 μl/ml) concentration showed the biggest change in algicidal activity;
there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their
integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5.
Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina. 相似文献