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。     

Production and chitinase-binding ability of lipo-chitopentaose nodulation factor

The penta-N-acetyl-chitopentaose 2 has been prepared by using recombinant E. coli strains harboring the nodC gene (encoding chitooligosaccharide synthase) from Azorhizobium caulinodans. Then, the deacetylase NodB removed the N-acetyl moiety from the nonreducing terminus of 2 to give tetra-N-acetyl-chitopentaose 3. N-Acylation of 3 with stearyl chloride was performed in DMF containing water and provided the corresponding lipo-chitopentaose nodulation factor 4. A binding chitinase assay indicated that 4 was much more stable than 3.     

Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm

A protein purification procedure was developed to efficiently and effectively purify the target enzyme, chitinase A1 of Bacillus circulans WL-12, from Escherichia coli DH5alpha carrying the chiA gene with its natural promoter in the plasmid pNTU110. Chitinase A1 was purified to apparent homogeneity from E. coli periplasm with a final recovery of 90.6%. Two main steps were included in this protein purification procedure, ammonium sulfate precipitation (40% saturation) and anion-exchange chromatography at pH 6.0 using Q Ceramic HyperD column. The yield of chitinase A1 was estimated at 95 microg/L. A polyclonal antibody against chitinase A1 was raised by immunizing BALB/c mice with chitinase A1 purified from E. coli DH5alpha(pNTU110). As indicated by Western blot analysis, a 3000-fold diluted antibody detected purified chitinase A1 from E. coli DH5alpha(pNTU110) in an amount of at least 1 ng and specifically detected chitinase A1 produced by B. circulans WL-12.     

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.     

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.     

Escherichia coli can produce recombinant chitinase in the soil to control the pathogenesis by Fusarium oxysporum without colonization

Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wildtype strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from 108 CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.     

苜蓿根瘤菌nodC蛋白在大肠杆菌中的过量生成和分离纯化

苜蓿根瘤菌（Ｒｈｉｚｏｂｉｕｍｍｅｌｉｌｏｔｉ）ｎｏｄＣ蛋白是结瘤基因ｎｏｄＣ编码的４３ｋＤ多肽（ＮｏｄＣ）。应用噬菌体Ｔ７ＲＮＡ聚合酶／启动子表达系统．ｐＴ７－５作为载体质粒．构建了带有ｎｏｄＣ基因的ＰＢＦ６克隆．经诱导在大肠杆菌ＪＡＫＥ中获得表达,过量生成ＮｏｄＣ,占细胞总蛋白量的５％。经细胞膜蛋白组份的分离,Ｂｉｏ－ｇｅｌ柱层析,ＳＤＳ－ＰＡＧＥ电泳等获得了比较纯化的ＮｏｄＣ。     

The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a functional exochitinase that plays a role in the chitin degradation during the molting process

The silkworm, Bombyx mori, has been recently demonstrated to contain a bacterial-type chitinase gene (BmChi-h) in addition to a well-characterized endochitinase gene (BmChitinase). The deduced amino acid sequence of BmChi-h showed extensive structural similarities with chitinases from bacteria such as Serratia marcescens chiA and baculoviruses (v-CHIA). Bacterial-type chitinase genes have not been found from any eukaryotes and viruses except for lepidopteran insects and lepidopteran baculoviruses. Thus, it was suggested that BmChi-h may be derived from a bacterial or baculovirus chitinase gene via horizontal gene transfer. In this report, we investigated the biological function of BmChi-h. Our enzymological study indicated that a chitinase encoded by BmChi-h has exo-type substrate preference, which is the same as S. marcescens chiA and v-CHIA, and different from BmChitinase, which has endo-type substrate preference. An immunohistochemical study revealed that BmChi-h localizes in the chitin-containing tissues during the molting stages, indicating that it plays a role in chitin degradation during molting. These results suggest that BmChi-h (exochitinase) and BmChitinase (endochitinase) may catalyze a native chitin by a concerted mechanism. Cloning and comparison of BmChi-h orthologues revealed that bacterial-type chitinase genes are highly conserved among lepidopteran insects, suggesting that the utilization of a bacterial-type chitinase during the molting process may be a general feature of lepidopteran insects.     

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.     

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.     

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.     

BmNPV chitinase gene deletion enhances foreign gene expression in a BmN cell system

Bombyx mori nucleopolyhedrovirus (BmNPV) is extensively being studied as an expression vector for heterologous gene expression in silkworm-derived cells as well as in the host larvae or pupae. BmNPV chitinase is necessary for liquefaction of the virus-infected host insect. The influence of chitinase on the efficiency of foreign gene expression was studied to provide a scientific basis for improving the BmNPV expression system. The BmNPV chitinase gene ( chiA ) was deleted and the expression level of the polyhedrin promoter controlling the lac Z gene in BmN cells was determined. Sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) showed that β-galactosidase accounted for approximately 6.9 and 7.7% of the total protein in BmN cells infected with the chiA deficient Bm lac Z⁺ chiA ⁻ at 3 and 4 days post infection, while the total protein was 3.2 and 4.2% in cells infected with Bm lac Z⁺. The relative β-galactosidase activities in Bm lac Z⁺ chiA ⁻-infected cells improved 2.33- and 1.56-fold compared to those of Bm lac Z⁺-infected cells at 3 and 4 days post infection. The results of the present study suggest that chitinase deletion could improve the lacZ expression level in the BmNPV-BmN cell expression system.     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.     

HaSNPV几丁质酶重组蛋白的表达、纯化和复性

棉铃虫单核衣壳核型多角体病毒（HaSNPV）是我国第一个商品病毒杀虫剂,具有使用安全、害虫不产生抗药性等优点,是一种很有发展潜力的生物农药。幼虫虫体受病毒感染后,HaSNPV几丁质酶在其液化过程中起了很大的作用,因此可以作为增效剂以显著提高细菌、病毒、真菌等微生物杀虫剂的毒力,并具有更高的安全性。将HaSNPV几丁质酶基因构建到原核表达载体pET28a中,经测序检验后转化至大肠杆菌Rosetta,然后以IPTG作为诱导剂,目标蛋白以包涵体的形式得以成功表达。在变性条件下,包涵体经镍 次氮基三乙酸(Ni-NTA)柱层析纯化,并以两种不同的方法进行复性,均可获得具有活性的HaSNPV几丁质酶。     

Differentiation of chitinase-active and non-chitinase-active subpopulations of a marine bacterium during chitin degradation

The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium Pseudoalteromonas sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the chiA promoter was used to evaluate chiA gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97-N-acetyl-beta-D-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of chiA expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase.