Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)_4–6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)₂ and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)₂ or GlcNAc, which are easily taken up by the cells.     

Is there a relationship between N‐acetyl‐β‐d‐glucosaminidase activity of Metarhizium anisopliae (Metschn.) Sorokin (Hyphomycetes) isolates from peridomestic areas in Central Brazil and larvicidal effect on Aedes aegypti (L.) (Diptera,Culicidae)?

Abstract:  The larvicidal effect of Metarhizium anisopliae (Metschn.) Sorokin (Hyphomycetes) isolated in peridomestic areas in Central Brazil was tested in Aedes aegypti (L.) (Dipt., Culicidae), which is worldwide the primary vector for the viruses that cause human dengue and yellow fever. Highest susceptibility of larvae was found after application of suspended ungerminated conidia. However, conidia, which were found on the larval cuticle and in the gut did not germinate in live or dead larvae. Mortality dropped when testing germinating conidia or supernatants, which originated from chitin-amended minimal medium (MM) inoculated conidia and cultures up to 72 h. Paralysis of larval movement was observed a few hours after application, especially of untreated conidia. Isolates showed a high variability of total protein production and N -acetyl- β - d -glucosaminidase activity after 48 and 72 h incubation in MM. No relationship between enzyme levels and insecticidal activity could be detected. The results indicate that toxic compounds emitted by ungerminated conidia on the cuticle or in the gut are involved in the activity of M. anisopliae against A. aegypti larvae.     

麻疯树几丁质酶性质的研究

为了评价麻疯树几丁质酶的性质,本研究采用不同pH的提取缓冲液提取麻疯树种子的几丁质酶,并比较分析了不同温度、不同反应时间对几丁质酶活性的影响。结果表明,在提取液pH为7．0时获得的几丁质酶活性最高;且在55℃时与底物胶体几丁质温育60min后可达最高酶活性。同时,本研究还提取麻疯树幼苗不同器官几丁质酶,并比较其比活力,结果表明,在叶中几丁质酶比活力最高,而根中比活力最低。实验结果为进一步研究麻疯树提供了理论依据。     

一株产几丁质酶菌株的筛选及其产酶条件的研究

采用平板透明圈法从土壤中分离筛选到一株产几丁质酶放线菌株L12,用250mL摇瓶发酵初筛和复筛,酶活力为0.63U/mL。通过产酶条件实验,初步确定了该菌株较适产酶培养基和摇瓶发酵条件。条件优化后,30℃、250mL摇瓶发酵48h,几丁质酶活力达到1.06U/mL。     

大白菜几丁质酶CHB4基因的表达研究

根据本研究组已克隆的大白菜ClassⅣ类几丁质酶基因序列设计引物,通过RT-PCR反应扩增得到该几丁质酶成熟肽基因CHB4,构建原核表达载体pET-CHB4,利用IPTG诱导表达并对诱导表达参数进行优化,SDS-PAGE分析表明,该基因表达的蛋白分子量为28 kD左右,其表达产物主要以包涵体的形式存在,25℃并没有改变表达蛋白的可溶性,而在pH9.5的培养基条件下,诱导表达产物的上清液中却有重组蛋白条带出现。以酵母菌为指示菌做抑菌圈实验,结果显示IPTG浓度为0.6 mmol/L时抑菌效果最明显。几丁质酶的活力测定结果表明,当IPTG浓度为0.6 mmol/L时,几丁质酶活力达到最大值2.8 U/mL。     

Characterization of chitinase from Streptomyces luridiscabiei U05 and its antagonist potential against fungal plant pathogens

Streptomyces luridiscabiei U05 was isolated from wheat rhizosphere. It produced chitinase, which showed in vitro antifungal properties. The crude enzyme inhibited the growth of Alternaria alternata, Fusarium oxysporum, F. solani, Botrytis cinerea, F. culmorum and Penicillium verrucosum. The chitinase enzyme of the molecular weight of 45 kDa was purified using affinity chromatography of chitin. Streptomyces luridiscabiei U05 produced different chitinolytic enzymes. The highest enzyme activity was observed with the use of 4‐MU‐(GlcNAc)_, which points to the presence of an β‐N‐acetylhexosaminidase. The optimum activity was obtained at 35–40°C and pH 7–8. The enzyme showed thermostability at 35–40°C during 240 min of preincubation and lost its activity at 50°C and 60°C in 60 min. The chitinase activity from S. luridiscabei U05 was strongly inhibited by Hg²⁺ and Pb²⁺ ions, and sodium dodecyl sulphate (SDS). The Ca²⁺, Cu²⁺ and Mg²⁺ ions stimulated the activity of the enzyme.     

Identification of chitinase mRNA in abscission zones from bean (Phaseolus vulgaris Red Kidney) during ethylene-induced abscission

Abstract. Total RNA was extracted from bean leaf abscission zones at different times after the induction of abscission by ethylene. The RNA was translated in the wheat germ system and the products analysed by SDS-PAGE. Products of molecular weight (raw) 42, 32 and 17 kD were seen to accumulate substantially during the induction. An attempt was made to establish that the mRNA species which produced the 32 kD product, which was coded for the ethylene-regulated enzyme chitinase. Mature chitinase (30 kD) was purifed from ethylene-treated abscission zones and used to raise monospecific antibodies in rabbits. These antibodies recognized the 32 kD product and mature chitinase. The 2 kD difference in molecular weight was due to the presence of the signal sequence which could be removed by microsomal membranes. Chitinase was also detected by enzymatic assay and immunoblotting of crude homogenates from ethylene-treated abscission zones. Chitinase appears to be ubiquitous in bean plants and probably does not have a direct role in abscission.     

Amino acid sequence of the N-terminal domain of yam (Dioscorea japonica) aerial tuber acidic chitinase. Evidence for the presence of a wheat germ agglutinin domain in matured acidic chitinase from unstressed tuber

The amino acid sequence of the N-terminal domain of acidic chitinase from unstressed aerial tuber was determined and proved the presence of an N-terminal domain in acidic chitinase. The amino acid sequence was determined on a pyroglutamylaminopeptidase-treated N-terminal fragment of V8 protease and on chymotryptic peptides of this fragment. The sequence determined revealed 8 residues deletion and 2 residues insertion as compared with the N-terminal domain of tobacco basic chitinase. The N-terminal domain determined showed a homology of 40% and 52% with the N-terminal domain of tobacco basic chitinase and wheat germ agglutinin, respectively.Abbreviations DABITC,4-N,N dimethylaminoazobenzene 4-isothiocyanate - PITC phenylisothiocyanate - Cm carboxymethyl - WGA wheat germ agglutinin - TFA trifluoroacetic acid - PGAP pyroglutamylaminopeptidase