Production and Secretion of a Recombinant Vibrio parahaemolyticus Chitinase by Escherichia coli and Its Purification from the Culture Medium

An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.     

一株拮抗香蕉枯萎病的内生细菌的分离及其几丁质酶基因信号肽的分泌活性分析

目的：旨在分离并选择一株香蕉内生细菌作为内生基因工程生防菌,并克隆其几丁质酶基因的信号肽序列。方法：从香蕉植株杆下部分离并选择了一株拮抗香蕉枯萎病且具有分泌几丁质酶能力的内生细菌,对该菌株进行了形态观察、生理生化测定和16S rDNA序列分析,克隆了其几丁质酶基因的编码序列并预测了其信号肽,构建了含有信号肽和不含信号肽的几丁质酶的表达菌株BL-chi1和BL-chi2。结果：结合形态观察、生理生化特征和16S rDNA序列比对分析确定该菌株为Klebsiella属,将该菌株命名为KKWB 5;BL-chi1和BL-chi2经IPTG诱导后,均表达了与预期蛋白大小一致的蛋白,同时BL-chi1诱导后的培养基上清中出现一条约45kDa的条带,而BL-chi2和空载体的BL-pET22b诱导后的培养基上清中均无此条带;几丁质水解试验发现,BL-chi1诱导后的培养基上清中的蛋白经浓缩和纯化后都能在几丁质平板上形成透明水解圈。结论：该几丁质酶的信号肽能被BL21（DE3）所识别,将几丁质酶分泌到培养基中,并且分泌的几丁质酶具有水解几丁质的生物学活性。内生菌KKWB-5的分离及其几丁质酶分泌信号肽序列的克隆为进一步构建内生工程菌来防治香蕉枯萎病打下了基础。     

昆虫杆状病毒几丁质酶及其应用研究进展

杆状病毒几丁质酶基因（chitinase,,ChiA）是晚期表达的非必需基因,高度保守。表达产物几丁质酶分为3个区：N-端信号肽区,中部酶活性区和C-末端酶内质网结合区。该酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的几丁质,促进虫体液化;作为组织蛋白酶原（pro-V-Cath）的分子伴侣,参与其加工和运输过程; 影响多角体的形成,并与细胞的裂解有关;还与病毒侵染机制相关联。杆状病毒ChiA与细菌ChiA源于共同的祖先,而昆虫ChiA则可能直接来自杆状病毒。在害虫生物防治中,杆状病毒ChiA可直接作为杀虫剂,或作为苏云金杆菌和杆状病毒等微生物杀虫剂的增效剂使用;杆状病毒ChiA可转入植物,获得具有持续杀虫及抗病活性的转基因植物;将杆状病毒ChiA的内质网定位序列删除、突变,或在病毒基因组中插入外源ChiA,重组病毒的杀虫活性增强。通过基因工程手段,删除病毒基因组ChiA或V-Cath,可改善杆状病毒表达系统对分泌蛋白和膜结合蛋白的表达。     

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.     

Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.     

一种新的抗真菌几丁质酶基因的分离及其植物表达载体的构建

以西瓜尖镰孢菌诱导、提纯的豇豆抗真菌 I类几丁质酶 N端前 1 0个氨基酸序列测定的基础上 ,设计合成了引物 ,运用 PCR等分子生物学技术 ,从豇豆基因组中分离克隆了该特异几丁质酶成熟蛋白基因 ,测定分析了其全序列。该新基因全长 894bp,无内含子 ;具 Aat I、Aat II、Bgl I、Dpn I、Dpn II、Eco R II、Hae I、Hae II、Hae III、Hinf I、Hpa II、Mae II、Mae III、Nba I、Oxa I和 Sst IV酶切位点 43个 ;豇豆、Vigna unguiculata、菜豆、豌豆、烟草、小麦、水稻的同源性依次递减。扩增克隆了菜豆几丁质酶信号肽基因 ,并将其与豇豆几丁质酶成熟蛋白基因连接 ,再与 p BI1 2 1重组 ,成功构建了特异几丁质酶基因的植物表达载体 ,为进一步培育抗真菌病转基因西瓜新品种打下了坚实基础。     

Expression and Export of Pseudomonas putida NTU-8 Creatinase by Escherichia coli Using the Chitinase Signal Sequence of Aeromonas hydrophila

The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Altermonas sp. strain O-7.

The gene encoding an extracellular chitinase from marine Alteromonas sp. strain O-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase of Alteromonas sp. strain O-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.     

Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin

A chitinase gene of Bacillus circulans WL-12 was cloned into Escherichia coli by transforming HB101 cells with a recombinant plasmid composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pKK223-3. DNA sequencing analysis revealed that the region necessary for the normal expression of chitinase activity contained one open reading frame of 2097 base pairs which codes for the precursor of chitinase A1. The precursor of chitinase A1 contained a long signal sequence of 41 amino acids with an extremely long N-terminal hydrophilic segment of 15 amino acids. Cloned chitinase produced in E. coli had at its N terminus an additional 8 amino acids that were not found in B. circulans mature chitinase A1. The N-terminal two-thirds of the deduced amino acid sequence of chitinase A1 showed a 33% amino acid match to chitinase A of Serratia marcescens. This region of chitinase A1 is immediately followed by tandemly repeating 95-amino acid segments that are 70% homologous to each other. Statistical analysis revealed that these repeating segments are homologous to the type III homology units of fibronectin, a multifunctional extracellular matrix and plasma protein of higher eukaryotes. This observation indicates that type III homology units originated prior to the emergence of eukaryotes and may be distributed in a wide range of organisms.     

Intracellular localization of a class IV chitinase from yam

Genomic DNA encoding a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves in previous research (Biosci. Biotechnol. Biochem., 68, 1508-1517 (2004)). But this chitinase had an additional sequence composed of eight amino acids (a C-terminal extension) at the C-terminal, compared with class IV chitianses from other plants. In order to clarify the role of this C-terminal extension in cellular localization, plants and suspension-cultured cells of Nicotiana tabacum were transformed with either the cloned yam class IV chitinase gene carrying the C-terminal extension or its truncated gene by the Agrobacterium-mediated method, and then their localization was investigated. The results suggest that the C-terminal extension of yam class IV chitinase plays a role as a targeting signal for plant vacuoles. This is the first report presenting the existence of vacuolar type class IV chitinase.     

Chitinase from Bacillus licheniformis DSM13: expression in Lactobacillus plantarum WCFS1 and biochemical characterisation

The gene chi, coding for a GH18 chitinase from the Gram-positive bacterium Bacillus licheniformis DSM13 (ATCC 14580), was cloned into the inducible lactobacillal expression vectors pSIP403 and pSIP409, derived from the sakacin-P operon of Lactobacillus sakei, and expressed in the host strain Lactobacillus plantarum WCFS1. Both the complete chi gene including the original bacillal signal sequence as well as the mature chi gene were compared, however, no extracellular chitinase activity was detected with any of the constructs. The chitinase gene was expressed intracellularly as an active enzyme with these different systems, at levels of approximately 5mg of recombinant protein per litre of cultivation medium. Results obtained for the two different expression vectors that only differ in the promoter sequence were well comparable. To further verify the suitability of this expression system, recombinant, His-tagged chitinase Chi was purified from cell extracts of L. plantarum and characterised. The monomeric 65-kDa enzyme can degrade both chitin and chitosan, and shows properties that are very similar to those reported for the native chitinase purified from other B. licheniformis isolates. It shows good thermostability (half lives of stability of 20 and 8.4 days at 37 and 50°C, respectively), and good stability in the pH range of 5-10. The results presented lead the way to overproduction of chitinase in a food-grade system, which is of interest for the food and feed industry.     

A class I chitinase from soybean seed coat.

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.     

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.     

Expression and secretion of Janthinobacterium lividumchitinase in Saccharomyces cerevisiae

The Janthinobacterium lividum chi69 chitinase gene linked to the Kluyveromyces lactis killer toxin secretion signal was ligated to the galactose-inducible CYC-GAL hybrid promoter of pEMBLyex4 and transferred directly into Saccharomyces cerevisiae DY-150. Exogenous chitinase activity assayed with 4-methylumbelliferyl--chitotrioside reached a maximum of 0.7 U/ml in the growth medium after 24 h galactose induction without any apparent deleterious effects on the yeast expression host.