Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca²⁺. However, Hg²⁺ and Ni⁺ ions strongly inhibited the enzyme. The enzyme produced, GlcN_2–4, were the major products from a soluble chitosan.     

Thermostable Chitosanase from Bacillus sp. Strain CK4: Cloning and Expression of the Gene and Characterization of the Enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80°C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.     

Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp.

A gene encoding a chitosanase (mschito) was cloned from Microbacterium sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase.     

Purification,Characterization, and Gene Analysis of Cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both β-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the β-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to β-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Thermostable chitosanase from Bacillus sp. Strain CK4: cloning and expression of the gene and characterization of the enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80 degrees C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.     

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34 805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50°C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl. Received: June 10, 2000 / Accepted: October 3, 2000     

Sequence analysis of the gene encoding a novel l-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases

The gene (ddc) encoding a novel enzyme, l-2,4-diaminobutyrate decarboxylase (DABA-DC; EC 4.1.1.-) in Acinetobacter baumannii was sequenced, and an open reading frame of 1,530 nucleotides was detected. The sequence of 20 N-terminal amino acids of purified DABA-DC and of its proteolytic peptide fragments coincided with those deduced from the nucleotide sequence determined. Comparison of the predicted amino acid sequence of the A. baumannii enzyme with those of other pyridoxal 5′-phosphate-dependent decarboxylases revealed significant similarity to the group II amino acid decarboxylases and conservation of the putative pyridoxal 5′-phosphate-binding domain. Received:20 February 1996 / Accepted 15 April 1996     

Identification of a fibrinolytic enzyme by <Emphasis Type="Italic">Bacillus vallismortis</Emphasis> and its potential as a bacteriolytic enzyme against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

A potent fibrinolytic enzyme-producing bacterium was isolated from the traditional Korean condiment Chungkook-jang and identified as Bacillus vallismortis Ace02. The extracellular fibrinolytic enzyme was purified with a 18% recovery of activity from supernatant cultures using CM-Sepharose column chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme was 757 kFU mg⁻¹. Its molecular mass was about 28 kDa and the initial amino acids of the N-terminal sequence were AQSVPYGVSQ. The full amino acid sequence of fibrinolytic enzyme Ace02 corresponded with bacteriolytic enzyme, L27, from Bacillus licheniformis, which has strong lytic activity against Streptococcus mutans, a major causative strain of dental caries. This suggests that the purified enzyme should be used for prevention of dental caries as well as being an effective thrombolytic agent.     

Molecular Characterization of a Novel Family-46 Chitosanase from Pseudomonas sp. A-01

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5–8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.     

Isolation, purification, and enzymatic characterization of extracellular chitosanase from marine bacterium Bacillus subtilis CH2

A Bacillus subtilis strain was isolated from the intestine of Sebastiscus marmoratus (scorpion fish) that was identified as Bacillus subtilis CH2 by morphological, biochemical, and genetic analyses. The chitosanase of Bacillus subtilis CH2 was best induced by fructose and not induced with chitosan, unlike other chitosanases. The strain was incubated in LB broth, and the chitosanase secreted into the medium was concentrated with ammonium sulfate precipitation and purified by gel permeation chromatography. The molecular mass of the purified chitosanase was detected as 29 kDa. The optimum pH and temperature of the purified chitosanase were 5.5 and 60°C, respectively. The purified chitosanase was continuously thermostable at 40°C. The specific acitivity of the purified chitosanase was 161 units/mg. The N-terminal amino acid sequence was analyzed for future study.     

Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene

A chitosanase produced constitutively by Bacillus sp. MET 1299 was purified by SP-Sephadex column chromatography. The molecular weight was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzyme activity was observed at a pH of 5.5 and temperature of 60 degrees C. The purified chitosanase showed high activity on 90% deacetylated colloidal chitosan and beta-glucan, but not on hydrolyzed colloidal chitin, CMC, or their derivatives. The N-terminal amino acid sequence of the enzyme was determined. The cloned full length gene, 1362 bp in size, encoded a single peptide of 453 amino acids and had a conserved amino acid sequence of glycosyl hydrolase family 8. A search of the cDNA sequence with NCBI BLAST showed homology with chitosanase of Bacillus sp. KTCC 0377BP and Bacillus sp. No. 7-M. The recombinant protein was expressed in Escherichia coli, purified using affinity chromatography and characterized.     

A prokaryotic sucrose synthase gene (susA) isolated from a filamentous nitrogen-fixing cyanobacterium encodes a protein similar to those of plants

Sucrose synthase (SS), a key enzyme in plant carbohydrate metabolism, has recently been isolated from Anabaena sp. strain PCC 7119, and biochemically characterized; two forms (SS-I and SS-II) were detected (Porchia et al. 1999, Planta 210: 34–40). The present study describes the first isolation and characterization of a prokaryotic SS gene, susA, encoding SS-II from that strain of Anabaena. A 7 kbp DNA fragment containing an open reading frame (EMBL accession number AJ010639) with about 30–40% amino acid identity with plant SSs was isolated from an Anabaena subgenomic library. The putative SS gene was demonstrated to encode an SS protein by expression in Escherichia coli. The biochemical properties of the recombinant enzyme were identical to those of the enzyme purified from the cyanobacterial cells. The deduced amino acid sequence of the Anabaena SS diverged from every plant SS reported. The occurrence of SS in cyanobacteria of different taxonomic groups was investigated. The enzyme occurs in several filamentous nitrogen-fixing cyanobacteria but not in two species of unicellular, non-diazotrophic cyanobacteria. Received: 5 January 2000 / Accepted: 7 March 2000     

Gene cloning and biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108

The DNA sequence of the thermostable chitosanase TCH-2 gene from Bacillus coagulans CK108 showed a 843-bp open reading frame that encodes a protein of 280 amino acids with a signal peptide corresponding to 32 kDa in size. The deduced amino acid sequence of the chitosanase from Bacillus coagulans CK108 has 61.6%, 48.0%, and 12.6% identities to those from Bacillus ehemensis, Bacillus circulans, and Bacillus subtilis, respectively. C-Terminal homology analysis shows that the enzyme belongs to the Cluster I group. The size of the gene was similar to those from mesophiles of the Cluster I group with regard to higher preference for codons ending in G or C. The recombinant chitosanase was electrophoretically purified to homogeneity by only two steps with column chromatography. The half-life of the enzyme was 40 min at 90 degrees C. The purified protein was also highly stable, retaining above 50% residual activities during treatment with denaturants such as urea (8 M) and guanidine x HCl (4 M) at 37 degrees C for 30 min. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, producing the tetramer as a major product.     

Characterization of the novel antifungal chitosanase PgChP and the encoding gene from Penicillium chrysogenum

The protein PgChP is a new chitosanase produced by Penicillium chrysogenum AS51D that showed antifungal activity against toxigenic molds. Two isoforms were found by SDS-PAGE in the purified extract of PgChP. After enzymatic deglycosylation, only the smaller isoform was observed by SDS-PAGE. Identical amino acid sequences were obtained from the two isoforms. Analysis of the molecular mass by electrospray ionization-mass spectrometry revealed six major peaks from 30 to 31 kDa that are related to different levels of glycosylation. The pgchp gene has 1,146 bp including four introns and an open reading frame encoding a protein of 304 amino acids. The translated open reading frame has a predicted mass of 32 kDa, with the first 21 amino acids comprising a signal peptide. Two N glycosylation consensus sequences are present in the protein sequence. The deduced sequence showed high identity with fungal chitosanases. A high level of catalytic activity on chitosan was observed. PgChP is the first chitosanase described from P. chrysogenum. Given that enzymes produced by this mold species are granted generally recognized as safe status, PgChP could be used as a food preservative against toxigenic molds and to obtain chitosan oligomers for food additives and nutraceuticals.     

Characterization of chitosanase of a deep biosphere Bacillus strain

A chitosanase of deep-biosphere Bacillus thuringiensis strain JAM-GG01 was purified. The optimal pH and temperature for the purified enzyme (Cho-GG) were about pH 6 and 60 °C, but Cho-GG was unexpectedly unstable under incubation at over 40 °C. This discrepancy between higher activity and lower stability in the same range of temperature was abolished by the addition of reaction products, chitotriose and chitotetraose. The Cho-GG gene was amplified by PCR and sequenced. The deduced amino acid sequence of Cho-GG showed more than 98% identity to those of other Bacillus enzymes belonging to GH family 8. Although Cho-GG did not show the definite characteristics of a sub-seafloor ectoenzyme, the thermal stability of many chitosanases of B. turingienesis and other related strains can be improved by adding chitotriose or chitotetraose.     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079     

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.     

Biodegradation of nylon oligomers

This mini-review is a compendium of the degradation of a man-made compound, 6-aminohexanoate-oligomer, in Flavobacterium strains. The results are summarized as follows: 1. Three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endotype 6-aminohexanoate-oligomer hydrolase (EIII) were responsible for degradation of the oligomers. 2. The genes coding these enzymes were located on pOAD2, one of three plasmids harbored in Flavobacterium sp. KI72, which comprised 45,519 bp. 3. The gene coding the EII′ protein (a protein having 88% homology with EII) and five IS6100 elements were identified on pOAD2. 4. The specific activity of EII was 200-fold higher than that of EII′. However, altering two amino acid residues in the EII′ enzyme enhanced the activity of EII′ to the same level as that of the EII enzyme. 5. The deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (encoding oligopeptide permease), ftsX (filamentation temperature sensitivity), penDE (isopenicillin N-acyltransferase) and rep (plasmid replication). 6. The EI and EII genes of Pseudomonas sp. NK87 (another nylon oligomer-degrading bacterium) were also located on plasmids. 7. Through selective cultivation using nylon oligomers as a sole source of carbon and nitrogen, two strains which initially had no metabolic activity for nylon oligomers, Flavobacterium sp. KI725 and Pseudomonas aeruginosa PAO1, were given the ability to degrade xenobiotic compounds. A molecular basis for the adaptation of microorganisms toward xenobiotic compounds was described. Received: 25 February 2000 / Received revision: 22 May 2000 / Accepted: 26 May 2000     

Molecular cloning and characterization of the gene encoding a fibrinolytic enzyme from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Strain A1

A fibrinolytic metalloprotease gene from Bacillus subtilis has been cloned in Escheridria coliXL1-Blue and the bacterial expressed enzyme was purified. The nucleotide sequence of the cloned fibrinolytic enzyme gene revealed a single open reading frame of 1023 bp coding for 341 amino acids (M _r 37708.21 Da). N-terminal amino acid sequencing of the fibrinolytic enzyme excreted from E. coli host cells revealed that the mature fibrinolytic enzyme consists of 288 amino acids (M _r 31391.1 Da). The deduced amino acid sequence showed significant homology with Erwina carotovora neutral metalloprotease and Serratia marcescens minor metalloprotease by 65 and 58% amino acid sequence identity, respectively. The protein showed significant alignments with the conserved domain of catalytic activity and the -helix domain in Bacillus anthracisthermolysis metalloprotease. The biochemical properties of the purified enzyme suggested that the enzyme is a fibrinolytic metalloprotease, which has optimal activity at pH 7.0 and 50 °C.