昆虫几丁质合成及其调控研究前沿

几丁质合成与降解是昆虫最重要的生理过程之一。本文根据国外和作者自己的研究,综述了昆虫几丁质合成及其调控研究进展。昆虫几丁质的生物合成通路始于海藻糖,终止于几丁质,其中共有8个酶参与。目前研究最多的为海藻糖酶和几丁质合成酶。昆虫存在2个海藻糖酶基因和2个几丁质合成酶基因。可溶性海藻糖酶基因对昆虫表皮的几丁质合成影响更大,而膜结合海藻糖酶基因则主要影响中肠的几丁质合成。几丁质合成酶A主要负责表皮和气管几丁质的合成,而几丁质合成酶B则负责中肠围食膜的几丁质合成。目前,昆虫几丁质合成的调控途径主要有两种:利用RNAi技术和几丁质合成抑制剂。     

昆虫几丁质合成酶及其抑制剂

几丁质合成酶(CS)是几丁质合成的关键酶,它具有3个结构域:结构域A、结构域B和结构域C,其中结构域B是催化域。根据氨基酸序列的差异,几丁质合成酶分为两类:CS-A及CS-B,分别在表皮及围食膜基质中催化合成几丁质。关于几丁质合成有2种假想模型。有多种抑制剂可以抑制几丁质的合成,其中核苷肽抗生素类及核苷磷酸类作用于CS的催化部位,是竞争性抑制剂,其它抑制剂的作用机理仍不明确。     

甲壳素酶学研究现状

本文介绍了甲壳素在生物合成和分解代谢过程中所涉及的相关酶,如甲壳素合成酶、甲壳素水解酶和其它相关酶,讨论了它们在分离纯化、结构鉴定、作用机制与模型、酶的固定化、基因工程以及应用等方面的研究现状和进展,对甲壳素的研究开发以及相关领域具有理论和实际意义 。     

Adsorption and reactions of chitinase and lysozyme on chitin

Summary Isotherms for adsorption of chitinase on chitin and lysozyme on chitin have been determined at two temperatures and rates of hydrolysis of chitin catalysed by these enzymes have been measured at three temperatures and at several enzyme concentrations for each. Ribonuclease, not an enzyme for chitin, and heat-denatured lysozyme and chitinase show reduced or no adsorption to this substrate.Initial hydrolysis rates of chitin by both enzymes are proportional to total enzyme concentrations in the range of concentrations studied. These kinetics cannot, however, be related to the adsorption isotherms because of the non-equilibrium nature of the isotherms.     

Detection and characterization of chitinases and other chitin-modifying enzymes

Multiple industrial and medical uses of chitin and its derivatives have been developed in recent years. The demand for enzymes with new or desirable properties continues to grow as additional uses of chitin, chitooligosaccharides, and chitosan become apparent. Microorganisms, the primary degraders of chitin in the environment, are a rich source of valuable chitin-modifying enzymes. This review summarizes many methods that can be used to isolate and characterize chitin-modifying enzymes including chitin depolymerases, chitodextrinases, chitin deacetylases, N-acetylglucosaminidases, chitin-binding proteins, and chitosanases. Chitin analogs, zymography, detection of reducing sugars, genomic library screening, chitooligosaccharide electrophoresis, degenerate PCR primer design, thin layer chromatography, and chitin-binding assays are discussed.     

Chitin metabolism

Recent studies on chitin metabolism in insect cuticle are reviewed. Differences in enzymes involved in both synthesis and degradation of chitin are discussed. Emphasis is put on the complexity of chitin degradation involving various enzymes. Evidence for the possible existence of an alternative pathway leading to the formation of chitin is introduced. The involvement of hormones in chitin metabolism is also briefly discussed.     

Increased Activity of Wall Hydrolytic Enzymes May Explain the Phenotype of Saccharomyces cerevisiae Fragile Mutants

Fragile mutants of Saccharomyces cerevisiae require osmotic stabilizers and lyse in hypotonic solutions. A single recessive mutation, srb1, is responsible for their phenotype, but the cause of cell lysis remains uncertain. We have analyzed three possible mechanisms for this behavior: comparative amounts of wall per cell; their chitin content; and the relative activity of wall hydrolytic enzymes activated by osmotic shock. We found normal amounts of wall and higher amounts of chitin in the fragile mutants. Determination of lytic enzymes by radiolabel of the reducing ends of wall polysaccharides gave results suggesting that fragile mutants produce increased amounts of stretch-activated wall hydrolytic enzymes, which may be responsible for their lysis in hypotonic media. These enzymes normally may play a role in cell wall growth and shaping. Received: 2 March 1998 / Accepted: 17 June 1998     

Overview of chitin metabolism enzymes in Manduca sexta: Identification,domain organization,phylogenetic analysis and gene expression

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.     

Substrate specificities of tobacco chitinases

Ten tobacco chitinases (1,4-N-acetyl-β-D-glucosaminide glycanhydrolase, EC 3.2.1.14) were purified from tobacco leaves hypersensitively reacting to tobacco mosaic virus. The 10 enzymes, which belong to five distinct structural classes of plant chitinases, were incubated with several potential substrates such as chitin, a β-1,4 N-acetyl-D-glucosamine (GlcNAc) polymer, chitosan (partially deacetylated chitin), chitin oligomers of variable length and bacterial cell wall. Tobacco chitinases are all endo-type enzymes that liberate oligomers from chitin and are capable of processing the chito-oligomers further at differential rates. Chitin reaction products were separated and quantified by HPLC and differential kinetics of oligomer accumulation and degradation were observed with the distinct classes of chitinases. Depending on the substrate to be hydrolysed, each isoform displayed a different spectrum of activity. For example, class I isoforms were the most active on chitin and (GlcNAc)_4–6 whereas class III basic isoforms were the most efficient in inducing bacterial lysis. Class V and class VI chitinases were shown to more readily hydrolyse chitin oligomers than the chitin polymer itself. Together, these data indicate that the 10 tobacco chitinases represent complementary enzymes which may have synergistic effects on their substrates. This paper discusses their implication in plant defense by attacking pathogen's structural components and in plant development by maturing signal molecules.     

Fungal chitinases and their biological role in the antagonism onto nematode eggs. A review

Chitin, the most abundant aminopolysaccharide in nature, is a rigid and resistant structural component that contributes to the mechanical strength of chitin-containing organisms. Chemically, it is a linear cationic heteropolysaccharide composed of N-acetyl-D-glucosamine and D-glucosamine units. The enzymatic degradation of chitin is performed by a chitinolytic system with synergistic and consecutive action. Diverse organisms (containing chitin or not) produce a great variety of chitinolytic enzymes with different specificities and catalytic properties. Their physiological roles involve nutrition, parasitism, chitin recycling, morphogenesis, and/or defense. Microorganisms, as the main environmental chitin degraders, constitute a very important natural source of chitinolytic enzymes. Nowadays, the most used method for pest and plant diseases control is the utilization of chemical agents, causative of significant environmental pollution. Social concern has generated the search for alternative control systems (i.e., biological control), which contribute to the generation of sustainable agricultural development. Interactions among the different organisms are the natural bases of biological control. Interest in chitinolytic enzymes in the field of biological control has arisen due to their possible involvement in antagonistic activity against pathogenic chitin-containing organisms. The absence of chitin in plants and vertebrate animals allows the consideration of safe and selective “target” molecules for control of chitin-containing pathogenic organisms. Fungi show appropriate characteristics as potential biological control agents of insects, fungi, and nematodes due to the production of fungal enzymes with antagonistic action. The antagonistic interactions between fungi and plant nematode parasites are among the most studied experimental models because of the high economic relevance. Fungi which target nematodes are known as nematophagous fungi. The nematode egg is the only structural element where the presence of chitin has been demonstrated. In spite of being one of the most resistant biological structures, eggs are susceptible to being attacked by egg-parasitic fungi. A combination of physical and chemical phenomena result in their complete destruction. The contribution of fungal chitinases to the in vitro rupture of the eggshell confirms their role as a pathogenic factor. Chitinases have been produced by traditional fermentation methods, which have been improved by optimizing the culture conditions for industrial processes. Although wild-type microorganisms constitute an alternative source of chitinolytic enzymes, the advances in molecular biology are allowing the genetic transformation of fungi to obtain strains with high capability as biocontrol agents. Simultaneously, a better understanding of rhizosphere interactions, additional to the discovery of new molecular biology tools, will allow the choosing of better alternatives for the biological control of nematodes in order to achieve an integrated management of the soil ecosystem.     

Two chitin synthases in Saccharomyces cerevisiae

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.     

Isolation of chitin-specific wheat oxidoreductases

Anionic peroxidase (IEP approximately 3.5) and oxalate oxidase (IEP approximately 7.0) were isolated from wheat seedlings using chitin. The strength of binding of enzymes with chitin depended on the degree of its acetylation and ionic strength of buffer. It was assumed that the acetyl groups of chitin are involved in sorption of enzymes on this biopolymer. The ability of anionic peroxidase and oxalate oxidase for sorption on chitin allows this biopolymer to be used for isolation of these proteins from plants. Cosorption of anionic peroxidase and oxalate oxidase on chitin suggests that these enzymes cooperate to ensure defensive response of wheat against chitin-containing pathogens.     

Studies on the Chitinolytie Enzymes of Black-koji Mold

In an attempt to separate the enzyme system participating in the decomposition of glycol chitin to constituent aminosugar, the purification of chitinase of Aspergillus niger was carried out by detemining both liquefying and saccharifying activities. Using fractionation with ammonium sulfate and column chromatography by hydroxylapatite, the chitinase system of the mold was separated into different enzyme fractions, which were required for the complete hydrolysis of glycol chitin. It was found that one of these enzymes caused a rapid decrease in viscosity of glycol chitin solution, another enzyme possessed N-acetyl-β-glucosaminidase activity upon N, N′-diacetylchitobiose and β-methyl-N-acetylglucosaminide, and that glycol chitin was decomposed to constituent aminosugar by a successive action of the two different enzymes.     

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus chitonophagus</Emphasis>

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80°C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.     

Purification and some properties of two forms of chitinase from mycelial cells of Mucor rouxii

Chitinase activity was measured in extracts of mycelial cells of Mucor rouxii as a function of the culture age. There was a peak of specific activity at the mid-exponential phase of growth (10 h), which paralleled chitin synthase activity. An additional peak of chitinase with higher specific activity was detected in 4 h cultures, which coincided with the onset of germination. Purification of chitinase activities from the cytoplasm revealed two enzymes, I and II, with different molecular mass and ionic charge. Antibodies induced with chitinase I did not cross-react with chitinase II. Both enzymes digested nascent chitin preferentially over preformed chitin, yielding diacetylchitobiose as the sole product of hydrolysis.     

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.     

Chitin-supplemented Foliar Application of Serratia marcescens GPS 5 Improves Control of Late Leaf Spot Disease of Groundnut by Activating Defence-related Enzymes

Chitinolytic Serratia marcescens GPS 5 and non‐chitinolytic Pseudomonas aeruginosa GSE 18, with and without supplementation of chitin, were tested for their ability to activate defence‐related enzymes in groundnut leaves. Thirty‐day‐old groundnut (cv. TMV 2) plants pretreated with GPS 5 and GSE 18 (with and without supplementation of 1% colloidal chitin) were challenge inoculated after 24 h with Phaeoisariopsis personata, the causal agent of late leaf spot (LLS) disease of groundnut. GPS 5 and GSE 18, applied as a prophylactic spray, reduced the lesion frequency by 23% and 67%, respectively, compared with control. Chitin supplementation had no effect on the control of LLS by GSE 18, unlike GPS 5, which upon chitin supplementation reduced the lesion frequency by 64%, compared with chitin alone. In a time course study the activities of chitinase, β‐1,3‐glucanase, peroxidase and phenylalanine ammonia lyase were determined for the different treatments. There was an enhanced activity of the four defence‐related enzymes with all the bacterial treatments when compared with phosphate buffer and colloidal chitin‐treated controls. In correlation to disease severity in bacterial treatments, chitin‐supplemented GSE 18 was similar to GSE 18, whereas chitin‐supplemented GPS 5 was much more effective than GPS 5, in activation of the defence‐related enzymes. The high levels of enzyme activities following chitin‐supplemented GPS 5 application continued up to the measured 13 days after pathogen inoculation.