Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.     

Induction and repression of a Streptomyces lividans chitinase gene promoter in response to various carbon sources

Induction and repression of a gene for chitinase (chiA) in Streptomyces lividans was investigated using a catechol 2,3-dioxygenase gene (xylE) as the reporter gene. Of various substrates examined, expression of the promoter (PchiA) was observed after a delay when colloidal chitin or small chitin-oligosaccharides were added to the medium. N-acetylglucosamine completely repressed the chiA promoter. The duration of the delay in expression of PchiA differed with the inducer used, with chitobiose inducing the activity most rapidly. The minimum concentration of chitobiose needed for induction was 1 microM. It appears, therefore, that an efficient inducer of the gene for chitinase in S. lividans is chitobiose.     

Identification and characterization of the gene cluster involved in chitin degradation in a marine bacterium, Alteromonas sp. strain O-7.

Alteromonas sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the chiA gene. The gene cluster consisted of three different open reading frames organized in the order chiD, cbp1, and chiA. The chiD, cbp1, and chiA genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The cbp1 and chiD genes were expressed in Escherichia coli, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when alpha-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc)(2) from chitin. The optimum temperature and pH of ChiD were 50 degrees C and 7.0, respectively.     

Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases.

The gene (chiD) encoding the precursor of chitinase D was found to be located immediately upstream of the chiA gene, encoding chitinase A1, which is a key enzyme in the chitinase system of Bacillus circulans WL-12. Sequencing analysis revealed that the deduced polypeptide encoded by the chiD gene was 488 amino acids long and the distance between the coding regions of the chiA and chiD genes was 103 bp. Remarkable similarity was observed between the N-terminal one-third of chitinase D and the C-terminal one-third of chitinase A1. The N-terminal 47-amino-acid segment (named ND) of chitinase D showed a 61.7% amino acid match with the C-terminal segment (CA) of chitinase A1. The following 95-amino-acid segment (R-D) of chitinase D showed 62.8 and 60.6% amino acid matches, respectively, to the previously reported type III-like repeating units R-1 and R-2 in chitinase A1, which were shown to be homologous to the fibronectin type III sequence. A 73-amino-acid segment (residues 247 to 319) located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitinases and class III higher-plant chitinases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha subunit. The evolutionary and functional meanings of these similarities are discussed.     

Cloning, expression, and characterization of a chitinase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9

The chitinolytic bacterium Aeromonas hydrophila strain SUWA-9, which was isolated from freshwater in Lake Suwa (Nagano Prefecture, Japan), produced several kinds of chitin-degrading enzymes. A gene coding for an endo-type chitinase (chiA) was isolated from SUWA-9. The chiA ORF encodes a polypeptide of 865 amino acid residues with a molecular mass of 91.6 kDa. The deduced amino acid sequence showed high similarity to those of bacterial chitinases classified into family 18 of glycosyl hydrolases. chiA was expressed in Escherichia coli and the recombinant chitinase (ChiA) was purified and examined. The enzyme hydrolyzed N-acetylchitooligomers from trimer to pentamer and produced monomer and dimer as a final product. It also reacted toward colloidal chitin and chitosan with a low degree of deacetylation. When cells of SUWA-9 were grown in the presence of colloidal chitin, a 60 kDa-truncated form of ChiA that had lost the C-terminal chitin-binding domain was secreted.     

Cloning and nucleotide sequence of the mycodextranase gene from Streptomyces sp. J-13-3

Mycodextranase (EC 3.2.1.61) is an alpha-glucanase that cleaves alpha-1,4-bonds of alternating alpha-1,3- and alpha-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.     

Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent phosphofructokinase.

The actinomycete Amycolatopsis methanolica employs a PPi-dependent phosphofructokinase (PPi-PFK) (EC 2.7.1.90) with biochemical characteristics similar to those of both ATP- and PPi-dependent enzymes during growth on glucose. A 2.3-kb PvuII fragment hybridizing to two oligonucleotides based on the amino-terminal amino acid sequence of PPi-PFK was isolated from a genomic library of A. methanolica. Nucleotide sequence analysis of this fragment revealed the presence of an open reading frame encoding a protein of 340 amino acids with a high degree of similarity to PFK proteins. Heterologous expression of this open reading frame in Escherichia coli gave rise to a unique 45-kDa protein displaying a high level of PPi-PFK activity. The open reading frame was therefore designated pfp, encoding the PPi-PFK of A. methanolica. Upstream and transcribed divergently from pfp, a partial open reading frame (aroA) similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes was identified. The partial open reading frame (chiA) downstream from pfp was similar to chitinase genes from Streptomyces species. A phylogenetic analysis of the ATP- and PPi-dependent proteins showed that PPi-PFK enzymes are monophyletic, suggesting that the two types of PFK evolved from a common ancestor.     

A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85 degrees C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.     

Characterization, expression in Streptomyces lividans, and processing of the amylase of Streptomyces griseus IMRU 3570: two different amylases are derived from the same gene by an intracellular processing mechanism.

Extracellular amylase in Streptomyces lividans was undetectable in starch-supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. griseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57-kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly.     

Cloning and Nucleotide Sequence of the Mycodextranase Gene from Streptomyces sp. J-13-3

Mycodextranase (EC 3.2.1.61) is an α-glucanase that cleaves α-1,4-bonds of alternating α-1,3- and α-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.     

Characterization of a chitinase gene (chiA) from Serratia marcescens BJL200 and one-step purification of the gene product

Abstract The nucleotide sequence of the chiA gene from Serratia marcescens strain BJL200 was determined. The gene was found to encode a protein of 563 amino acid residues, with a typical N-terminal signal peptide of 23 residues, that is cleaved off during export. The gene exhibited striking differences with two previously characterized chiA genes of S. marcescens in the region corresponding to amino acid residues 410–467 of the gene product. Periplasmic fractions of an Escherichia coli strain harbouring the cloned gene were used as starting material for the development of a fast, one-step purification protocol for the chitinase that is based on hydrophobic interaction chromatography.     

Hyperexpression and analysis of choB encoding cholesterol oxidase of Brevibacterium sterolicum in Escherichia coli and Streptomyces lividans.

We examined the expression of choB, encoding cholesterol oxidase of Brevibacterium sterolicum ATCC 21387, in Escherichia coli JM105 and Streptomyces lividans TK23 using various deletion DNA fragments within the 5'-flanking region. The enzyme activity could be detected intracellularly in E. coli only when the 5'-flanking region was reduced to less than 256-bp and choB was transcribed by the lac promoter. A large amount of the enzyme were produced as inactive inclusion bodies when ChoB protein was fused with the NH2-terminal portion of LacZ protein. In contrast, choB with more than 256-bp of the 5'-flanking region was efficiently expressed in S. lividans TK23, and about 85 times as much of the active enzyme (170 U/ml) was secreted into the culture filtrate as with B. sterolicum in flask culture. These results suggest that the promoter of choB exist within 256-bp of the 5'-flanking region and can be efficiently recognized by the RNA polymerase of S. lividans. The characteristics of the enzyme purified from the culture filtrate of the S. lividans transformant and that of B. sterolicum were identical although the NH2-terminal amino acid sequence of the enzyme from the S. lividans transformant was 6 amino acids shorter than that from B. sterolicum.     

Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control

A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.     

Cloning and primary structure of the chiA gene from Aeromonas caviae.

The chiA gene from Aeromonas caviae encodes an extracellular chitinase, 865 amino acids long, that shows a high degree of similarity to chitinase A of Serratia marcescens. Expression in Escherichia coli yielded an enzymatically active protein from which a leader sequence was removed, presumably during transport of the enzyme across the cell membrane.     

一株Sanguibacter sp.C4产几丁质酶基因的克隆与表达

Chi58是Sanguibacter sp．strain C4产生的一种胞外几丁质酶。通过chiA的特异性PCR引物探测到菌株C4中存在几丁质酶,并将扩增到的几丁质酶基因片段（chiA-F）克隆、测序后,提交GenBank数据库进行同源性搜索。对从GenBank中获得的高同源性序列进行比对,并根据保守区域设计2对PCR引物进行嵌套PCR,扩增出Chi58基因的开放阅读框（ORF）。测序结果表明该酶的ORF由1692个核苷酸组成,编码563个氨基酸,在N端有23个氨基酸的信号肽,其成熟蛋白的分子量应为58．544kDa。对其推导氨基酸的序列分析表明Chi58与沙雷氏菌的几丁质酶（如徂）有高度同源性（88．9％-99．6％）,其结构主要包括信号肽序列、PKD结构域和18家族糖苷水解酶结构域。将该基因克隆到pET32a（＋）载体构建重组质粒pChi58,转入大肠杆菌BL-21（DE3）进行融合表达。经IPTG诱导后,可见分子量约81．1kDa的融合蛋白的表达。     

Isolation of a chitinase overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Streptomyces peucetius, producer of the antitumor anthracycline antibiotic daunorubicin, was mutagenized, and mutants defective in daunorubicin biosynthesis were screened. One mutant (SPVI), which failed to produce daunorubicin, was found to overproduce an extracellular chitinase. Time course analyses of chitinase production and of the extracellular protein profile showed that the increase in activity is due to increased synthesis of the enzyme protein. The production of chitinase in SPVI was repressed by glucose as in the case of wild-type S. peucetius. PFGE analysis of VspI restriction fragments of S. peucetius and SPVI showed that there was no major alteration in the mutant genome. The hybridization pattern of S. peucetius and SPVI genomic DNA digested with various restriction enzymes was identical when probed with dnrUVJI genes of the S. peucetius daunorubicin cluster and chiA of Streptomyces lividans 66. The possible step affected in the daunorubicin biosynthetic pathway could be a polyketide synthase, since aklanonic acid, the earliest detectable intermediate in the daunorubicin pathway, was not synthesized in SPVI.