Production and Isolation of Levan by Use of Levansucrase Immobilized on the Ceramic Support SM-10

The complete amino acid sequence of rye seed chitinase-a (RSC-a) has been analyzed. RSC-a was cleaved with cyanogen bromide and the resulting three fragments, CB1, CB2, and CB3, were separated by gel filtration. The amino acids of the N-terminal fragment CB1 were sequenced by analyzing the peptides produced by digestion with trypsin, lysylendopeptidase, or pepsin of reduced S-carboxymethyl ated or S-aminoethylated CB1. The sequences of fragments CB2 and CB3 were established by sequencing the tryptic peptides from reduced S-carboxymethylated CB2 and CB3, and by aligning them with the sequence of rye seed chitinase-c (RSC-c) to maximize sequence homology. The complete amino acid sequence of RSC-a was established by connecting these three fragments.

RSC-a consists of 302 amino acid residues including hydroxyproline residues, and has a molecular mass of 31,722 Da. RSC-a is basic protein with a cysteine-rich amino terminal domain, indicating that this enzyme belongs to class I chitinases. The amino acid sequence of RSC-a showed that the sequence from Gly60 to C-terminal Ala302 in this enzyme corresponds to that of RSC-c belonging to class II chitinases with 92% identity, and that RSC-a has high similarity to other plant class I chitinases but a longer hinge region and an extra disulfide bond.     

Cloning, sequencing, and expression of the tulip bulb chitinase-1 cDNA

A cDNA encoding tulip bulb chitinase-1 (TBC-1) was cloned using a combination of immunoscreening from a lambda ZAP cDNA library with anti-TBC-1 antiserum and the 5' rapid amplification of cDNA end (RACE) method, and sequenced. The cDNA consists of 1,106 nucleotides and included an open reading frame encoding a polypeptide of 314 amino acids. Comparison of the deduced amino acid sequence and the determined protein sequence indicated the presence of a signal peptide and an extra peptide composed of 26 and 13 amino acids at the N- and C-termini, respectively. The deduced sequence of TBC-1 had 10-20% and 63% sequence similarities to plant class III chitinases and gladiolus bulb class IIIb chitinase (GBC-a), respectively. The cDNA encoding mature TBC-1 was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant TBC-1 (rTBC-1) expressed in E. coli was purified by gel filtration followed by ion-exchange chromatography. Specific activity of the rTBC-1 was almost same as the authentic TBC-1 toward glycolchitin. This is the first report on the cDNA cloning of a class III chitinase having C-terminal extra peptide.     

Purification, characterization, and cDNA cloning of rice class III chitinase

Several chitinases were expressed in a rice cell suspension culture and detected in the medium. One of them, designated as RCB4, was isolated 248 fold from the culture filtrate to homogeneity by 70% ammonium sulfate precipitation, DEAE-cellulose, CM-cellulose, Sephadex G-75 column chromatography, and native gel slicing. RCB4 had a molecular mass of 32 kDa by SDS-PAGE. The optimum temperature was 40 degrees C, and 96% of its activity still remained at 60 degrees C. The optimum pH was 4, and 95% of its activity was maintained at pH 2. Using a substrate (GlcNAc)6, the Km and Vmax values of RCB4 were 0.53 mM and 11.1 mM/min, respectively. The N-terminal and internal amino acid sequences of RCB4 were determined to be VNSNLFRDYIGA and MALWA, respectively. A cDNA (C12523) clone that contained the N-terminal and internal amino acid sequences of RCB4 was obtained, sequenced, and renamed RCB41. RCB41 encoded 307 amino acid protein with a signal peptide of 25 amino acids and showed a 45% similarity to gladiolus chitinase GBC-a, one of the class III chitinase family. The expression of RCB4l in E. coli showed that RCB41 encodes a chitinase.     

Conversion of α-Terpineol to 8,9-Epoxy-p-menthan-1-ol

The complete amino acid sequence of pokeweed lectin-B (PL-B) has been analyzed by first sequencing seven lysylendopeptidase peptides derived from the reduced and S-pyridylethylated PL-B and then connecting them by analyzing the arginylendopeptidase peptides from the reduced and S-carboxymethylated PL-B. PL-B consists of 295 amino acid residues and two oligosaccharides linked to Asn96 and Asnl39, and has a molecular mass of 34,493 Da. PL-B is composed of seven repetitive chitin-binding domains having 48–79% sequence homology with each other. Twelve amino acid residues including eight cysteine residues in these domains are absolutely conserved in all other chitin-binding domains of plant lectins and class I chitinases. Also, it was strongly suggested that the extremely high hemagglutinating and mitogenic activities of PL-B may be ascribed to its seven-domain structure.     

Purification, characterization, and molecular cloning of a chitinase from the seeds of Benincasa hispida

A chitinase was purified from the seeds of Benincasa hispida, a medicinal plant also called white gourd, and a member of the Cucurbitaceae family. Purification was done by using a procedure consisting of only two fractionation steps: an acid denaturation step followed by ion-exchange chromatography. The sequence of the N-terminal forty amino acid residues was analyzed and the sequence indicated that the enzyme is a class III chitinase. The enzyme, which is a basic chitinase, is one of at least five chitinases detected in the seed extract of B. hispida. Like other class III chitinases, this enzyme also has lysozyme activity. A genomic clone of the gene encoding the enzyme was isolated and sequenced. The gene has the potential to encode a protein of 301 amino acid residues. The deduced amino acid sequence of the protein, as expected from the N-terminal amino acid sequence, shares high degrees of similarity with other class III chitinases.     

豇豆几丁质酶N端序列测定及与其它植物的比较

通过自动 Edman降解程序 ,测定了经诱导、纯化的豇豆几丁质酶 N端 1 0个氨基酸的序列 ,并将该序列与其它植物几丁质酶 N端相应部分的氨基酸序列进行了比较分析。结果表明 ,该豇豆 ( Vigna sesquipedalis)几丁质酶 N端 1 0个氨基酸的序列为 EQCGSQAGGA,与 类几丁质酶同一部分同感序列同源性高达 1 0 0 % ;而与 、 及 类几丁质酶的相应序列均无同源性。结合考虑此酶的等电点 ( 8.3)及分子量 ( 33k D) ,可推测该豇豆几丁质酶属于 类几丁质酶。其 N端序列的高度保守性提示 ,该段序列可作为 类几丁质酶的一段主要特征序列 ,并可据其合成核酸探针 ,以分离、克隆其它 类几丁质酶编码基因。     

Amino acid sequence of chitinase from Streptomyces erythraeus

The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.     

Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco

Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.     

Characterzation of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variantts11

To characterize the acidic endochitinase EP3, able to rescue somatic embryos of the carrot cell linets11, the enzyme was purified from the medium of wild-type suspension cultures. Peptide sequences, deduced amino acid sequences of corresponding PCR-generated cDNA clones, serological relation and biochemical properties showed that there were at least five closely related chitinases, four of which could be identified as class IV EP3 chitinases with an apparent size of 30 kDa. Two other proteins were identified as a serologically related class I acidic chitinase (DcChitI) of 34 kDa, and a serologically unrelated 29 kDa class II acidic chitinase (DcChitII), respectively. Additional cDNA sequences, Western and Southern analysis showed the presence of a least two, but possibly more, highly homologous class IV EP3 genes in the carrot genome. Two class IV EP3 chitinases were tested and found to be able to increase the number ofts11 globular embryos formed under non-permissive conditions. One of the class IV EP3 chitinases as well as the class I chitinase DcChitI promoted the transition from globular to heart-stagets11 embryos. The class II endochitinase and a heterologous class IV chitinase from sugar-beet were not active onts11. This suggests that there are differences in the specificity of chitinases in terms of their effect on plant somatic embryos.     

Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid

The C-terminal propeptide of tobacco (Nicotiana tabacum) chitinase A has been shown to be necessary and sufficient for targeting of chitinases to the plant vacuole. The sequence specificity of this vacuolar targeting peptide (VTP) has now been analysed using transient expression of chitinases in Nicotiana plumbaginifolia protoplasts. An extracellular cucumber chitinase, previously used as a secreted reporter protein in transgenic tobacco, was also secreted into the incubation medium by the transiently transformed protoplasts. Addition of six to seven amino acids at the C-terminus to generate the VTP of tobacco chitinase A were sufficient to cause retention of most of the cucumber chitinase within the protoplasts. The chitinase A itself, as well as a mutant lacking the N-terminal chitin-binding domain, were retained to 80% in the protoplasts when low concentrations of the plasmid were used in the transient expression system. At high concentrations of plasmid, causing high levels of transiently expressed chitinase, retention was reduced, indicating saturation of the sorting system. Deletion of the C-terminal methionine did not affect the intracellular location, but deletion of even a single internal amino acid of the VTP caused predominantly secretion of tobacco chitinase A. In contrast, exchanges of amino acids in the VTP as well as substitution of the VTP with random sequences had intermediary effects that covered the whole range from retention to secretion. The results suggest that the sorting system responsible for the diversion of secretory proteins to the vacuole has a low specificity for the sequence of C-terminal targeting peptides, and that sequence changes in the VTP allow a gradual transition from vacuolar retention to secretion.     

Papaya (Carica papaya) Lysozyme Is a Member of the Family 19 (Basic, Class II) Chitinases

The most comprehensive studies on a plant lysozyme (EC 3.2.1.17) are those on the enzyme from papaya (Carica papaya) latex, published in 1967 and 1969. However, the N-terminal amino acid sequence of five amino acid sequence of this enzyme, determined by manual Edman degradation, did not allow assignment to any of the much later-classified families of glycosyl hydrolases. N-Terminal sequence analysis of 22 residues of papaya lysozyme now shows unambiguously that the enzyme belongs to the family 19 chitinases. It has properties similar to those of basic class I chitinases with lysozyme activity, such as cleavage specificity at the C-1 of N-acetylmuramic acid with inversion of configuration, but as it lacks an N-terminal hevein domain, it should be classified as a class II chitinase. Received: 3 February 1999 / Accepted 25 July 1999     

Molecular Cloning of a Genomic DNA Encoding Yam Class IV Chitinase

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.     

The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase.

The complete amino acid sequence of acidic chitinase from yam (Dioscorea japonica) aerial tubers was determined. The protein is composed of a single polypeptide chain of 250 amino acid residues and has a calculated molecular mass of 27,890 Da. There is an NH2-terminal domain, a hinge region, and a main structure, typical for class I chitinases (Shinshi, H., Neuhaus, J.-M., Ryals, J., and Meins, F., Jr. (1990) Plant Mol. Biol. 14, 357-368). We have obtained the first evidence for an acidic class I chitinase. Comparison with sequences of other class I chitinases revealed approximately 40% sequence similarity, a value lower than that for other class I chitinases (70-80%). We assume that there is a local conformational change in the molecule; cysteine residues that probably form disulfide bonds are completely conserved, with the exception of Cys-178. The difference in structure between this chitinase and other basic class I chitinases suggests that acidic and basic isoforms should be grouped into subclasses; this protein is an ethylene- or a pathogen-independent chitinase produced by a gene that is inherent in the tuber.     

棉铃虫核型多角体病毒几丁质酶基因及杆状病毒 几丁质酶基因的分子进化

用PCR方法扩增了棉铃虫Helicoverpa armigera单粒包埋型核型多角体病毒（HaSNPV）几丁质酶基因,测定了基因编码区的核苷酸全序列。基因编码区全长1.713 bp,可编码570个氨基酸残基组成的多肽,预计分子量为63.6 kD。将所推导的HaSNPV几丁质酶氨基酸序列与其它已知杆状病毒几丁质酶氨基酸序列进行联配比较,结果表明HaSNPV 与谷实夜蛾H.zea单粒包埋型核型多角体病毒（HzSNPV）的氨基酸序列非常相似,同源性高达90.7%,与苜蓿丫纹夜蛾Autographa californica多粒包埋型核型多角体病毒（AcMNPV）、家蚕Bombyx mori核型多角体病毒（BmNPV）、美国白蛾Hyphantria cunea核型多角体病毒（HcNPV）、舞毒蛾Lymantria dispar多粒包埋型核型多角体病毒（LdMNPV）、黄杉毒蛾Orgyia pseudotsugata多粒包埋型核型多角体病毒（OpMNPV）和云杉卷叶蛾Choristoneura fumiferana核型多角体病毒（CfMNPV）氨基酸序列同源性分别为64.4%、64.9%、64.2%、62.9%、66.2%和61.5%。根据氨基酸序列用PC＼GENE程序绘制已知杆状病毒几丁质酶的分子系统树,并与杆状病毒中最为保守的多角体蛋白基因系统树作了比较,结果表明几丁质酶基因和多角体蛋白基因的进化速率是不尽相同的。     

Characterization of two antifungal endochitinases from barley grain

A basic chitinase (chitinase T, EC 3.2.1.14, molecular mass 33 kDa, pI 9.8) was isolated and compared with a previously described chitinase (chitinase C, molecular mass 28 kDa, pI 9.7). The two chitinases were isolated in homogeneous form from barley ( Hordeum vulgare L.) Bomi mutant 1508 grains either by two cation exchange steps or by one affinity step followed by cation exchange. Both chitinases are endochitinases with specific activities of 168 and 54 nkat (mg protein)⁻¹ for chitinase T and chitinase C, respectively. Both inhibit the growth of Trichoderma viride efficiently. The lysozyme activity of both chitinases is 10⁴ times lower than that of hen egg-white lysozyme as measured by lysis of cell walls of Micrococcus lysodeikticus . The amino acid composition and two partial amino acid sequences of chitinase T were determined. A 23 residue sequence of the N-terminal domain of chitinase T, which was not present in chitinase C, showed 73% identity with domain B of wheat germ lectin and 65% identity with the N-terminal domain of an endochitinase from bean leaves (deduced from cDNA). A 9 amino acid sequence of a cyanogen bromide fragment of chitinase T was identical with a cDNA deduced sequence of a barley aleurone endochitinase but differed in one residue from chitinase C. Generally, the two grain chitinases have physico-chemical and enzymatic properties similar to the plant leaf chitinases characterized. Both chitinases are localized in the aleurone layer and starchy endosperm of developing and germinating grain, but not in the embryo. The appearance of chitinases T and C at a late state of grain development suggests a role for these enzymes as a defense against fungi in the quiescent and germinating grain.     

Three Extracellular Chitinases in Suspension-cultured Rice Cells Elicited by N-Acetylchitooligosaccharides

In rice suspension culture, a large part (about 90% of total activity in the culture) of the chitinase activity was found in the medium. Two extracellular chitinases (which we named RCH-A and -B) were separated from the cell suspension by DEAE-cellulofìne column chromatography. When cells were treated with N-acetylchitooligosaccharides (chitin oligosaccharides) for 3 days, extracellular chitinase activity increased about 3-fold over the control culture. After the treatment, another extracellular chitinase (named RCH-C) appeared in addition to increases in the levels of RCH-A and -B. Partial amino acid sequences of these enzymes indicated that RCH-A (33.5 kDa) and -B (34kDa) were class Ib chitinases but RCH-C (27kDa) was a class III chitinase. RCH-A and -B were capable of actively degrading water-insoluble chitin with high affinities, while RCH-C had less affinity for the substrate. However, when a water-soluble chitin derivative, 6–O-hydroxyethylchitin (glycolchitin) was used, RCH-C as well as RCH-A and -B degraded actively with a high affinity. A synergistic effect was observed when these three chitinases acted simultaneously in the hydrolysis of chitin.     

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I–IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I β-1,3-glucanase against Fusarium solani germlings.     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

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.