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1.
Biochimie ; 90(3): 525-33, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17988780

ABSTRACT

Three mutanase (alpha-1,3-glucanase)-producing microorganisms isolated from soil samples were identified as a relatives of Paenibacillus. A mutanase was purified to homogeneity from cultures of each, and the molecular masses of the purified enzymes were approximately 132, 141, and 141kDa, respectively. The corresponding three genes for mutanases were cloned by PCR using primers designed from each N-terminal amino acid sequence. Another mutanase-like gene from one strain was also cloned by PCR using primers designed from conserved amino acid sequences among known mutanases. Consequently, four mutanase-like genes were sequenced. The genes contained long open reading frames of 3411 to 3915bp encoding 1136 to 1304 amino acids. The deduced amino acid sequences of the mutanases showed relatively high similarity to those of a mutanase (E16590) from Bacillus sp. RM1 with 46.9% to 73.2% identity and an alpha-1,3-glucanase (AB248056) from Bacillus circulans KA-304 with 46.7% to 70.4% identity. Phylogenetic analysis based on the amino acid sequences of the enzymes showed bacterial mutanases form a new family between fungal mutanases (GH family 71) and Streptomycetes mycodextranases (GH family 87).


Subject(s)
Bacteria/enzymology , Glycoside Hydrolases/classification , Glycoside Hydrolases/genetics , Amino Acid Sequence , Bacteria/classification , Glycoside Hydrolases/chemistry , Molecular Sequence Data , Mutation , Phylogeny , Protein Structure, Tertiary , Sequence Analysis, Protein , Sequence Homology, Amino Acid
2.
J Biotechnol ; 129(3): 406-14, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17368606

ABSTRACT

Endoglucanase (Egl)-producing bacteria from soil samples were screened using insoluble cellulosic substrates as sole carbon sources at alkaline pH (pH 9-10). Four Egls with Avicelase activity at alkaline pH were found in the culture broth of each isolate. The Egl genes of the isolates (all Paenibacillus spp.) were shotgun cloned and sequenced-all had a 1752bp open reading frame (584 amino acids) with a putative signal sequence (33 amino acids), and encoded mature enzymes of 551 amino acids (58,360-58,672Da). The mature enzymes showed a high degree of similarity to each other (>93% identity), with the next closest similarity to Egl3a of a patented strain of Paenibacillus lautus NCIMB 40250 (81.5-87.3% identity). These enzymes showed low similarity to other known Egls with less than 50% identity. A representative recombinant enzyme degraded lichenan, carboxymethylcellulose (CMC), glucomannan, acid or alkaline swollen celluloses, and microcrystalline cellulose (Avicel). The optimal pH and temperature of the recombinant enzyme for degrading CMC and Avicel were pH 6.0-8.5 and 45-55 degrees C, respectively. Egls belong to glycoside hydrolase family 5 and form a distinct clan based on the phylogenetic analysis of their amino acid sequences.


Subject(s)
Bacteria/enzymology , Glycoside Hydrolases/genetics , Phylogeny , Amino Acid Sequence , Bacteria/genetics , Base Sequence , Cloning, Molecular , DNA Primers , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Soil Microbiology , Substrate Specificity , Temperature
3.
Extremophiles ; 8(3): 229-35, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15022105

ABSTRACT

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 degrees 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.


Subject(s)
Bacillus/enzymology , Bacillus/genetics , Serine Endopeptidases/genetics , Subtilisins/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Genes, Bacterial , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Molecular Weight , Multigene Family , Open Reading Frames , Phylogeny , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Subtilisins/chemistry , Subtilisins/metabolism
4.
Biochim Biophys Acta ; 1624(1-3): 109-14, 2003 Dec 05.
Article in English | MEDLINE | ID: mdl-14642820

ABSTRACT

A high-molecular-mass subtilisin was found in culture broth of the alkaliphilic Bacillus sp. strain KSM-KP43. The gene encoding the enzyme (FT protease) was determined using a mixed primer designed from the N-terminal amino acid (aa) sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of a 2427-bp open reading frame (ORF) that encoded a putative prepro-peptide (152 aa) and a mature enzyme (656 aa; 68,506 Da). The deduced aa of the mature enzyme revealed a moderate homology to a subtilisin-type proteinase from Bacillus halodurans and a minor extracellular protease, Vpr, from Bacillus subtilis with 64% and 57% identity, respectively. The molecular mass of the purified recombinant FT protease was approximately 72 kDa as judged by both SDS-polyacrylamide gel electrophoresis (PAGE) and gel filtration. FT protease showed maximal activity toward glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide at pH 10.5 and at 45 degrees C. The enzyme was rapidly inactivated by incubation over 45 degrees C for 15 min at both pH 7 and 10. Calcium ions were slightly protective for thermoinactivation of the enzyme.


Subject(s)
Bacillus/chemistry , Subtilisin/genetics , Amino Acid Sequence , Base Sequence , Hydrogen-Ion Concentration , Molecular Sequence Data , Substrate Specificity , Subtilisin/chemistry , Subtilisin/metabolism , Temperature
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