Armillaria gallica is a facultative parasitic fungus which is the only nutrient source of Gastrodia elata during its cultivation.Chitinase,as a glycosidic hydrolytic enzyme,plays an important role in the growth,development,stress tolerance and symbiotic signal transduction of A. gallica. There were 22 chitinase genes in A. gallica. Bioinformatics analysis of amino acid sequence of these chitinase genes revealed that 12 chitinase genes contained glycosidase 18 family( GH18) domain. Chitinase amino acid sequences of A. gallica,A. ostoyae,G. elata,Saccharomyces cerevisiae and Trichoderma harzianum were analyzed byclustering trees,so as to further predict the gene function of chitinase in A. gallica. Induction of A. gallica branching with strigolactone analogue GR24,high-throughput sequencing technology based on the induction of branch group( MHJ1),uninduced branch group( MHJ2) and blank control group( MHJ3) is used to detect the expression quantity,the transcription level data of 22 chitinase genes were obtained and the heat map was generated for expression pattern analysis. It was found that 8 genes may be involved in physiological processes such as A. gallica branching,cell wall degradation and remodeling. In this paper,the function of chitinase gene in A. gallica was just preliminarily analyzed and predicted.
Amino Acid Sequence ; Armillaria ; Chitinases ; Computational Biology ; Trichoderma

A novel chitinolytic and chitosanolytic bacterium, Sphingomonas sp. CJ-5, has been isolated and characterized. It secretes both chitinase and chitosanase into surrounding medium in response to chitin or chitosan induction. To characterize the enzymes, both chitinase and chitosanase were purified by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-Sepharose Fast Flow. SDS-PAGE analysis demonstrated molecular masses of chitinase and chitosanase were 230 kDa and 45 kDa respectively. The optimum hydrolysis conditions for chitinase were about pH 7.0 and 36 degrees C, and these for chitosanase were pH 6.5 and 56 degrees C, respectively. Both enzymes were quite stable up to 45 degrees C for one hour at pH 5~8. These results show that CJ-5 may have potential for industrial application particularly in recycling of chitin wastes.
Chitinases ; metabolism ; Enzyme Stability ; Fermentation ; Glycoside Hydrolases ; metabolism ; Hydrogen-Ion Concentration ; Sphingomonas ; enzymology

The importance of chitinases in the physiological and developmental processes of fungi and insects makes themselves and their inhibitors important targets for biological pesticides. A chitinase was isolated from Bombyx mori and purified to electrophoretic homogeneity by ammonium sulfate precipitation and Sephadex G-150 column chromatography. The molecular mass was estimated to be about 88 kDa by SDS-PAGE, while the K(m) was calculated to be 22.3 micromol/L. Moveover, the optimal reaction temperature was 45 degrees C, and the optimum pH was 6.0. The effect of metal ions and organic reagents on chitinase activity was investigated. The activity was enhanced by high concentration of Mn2+, while was strongly inhibited by Cu2+ and SDS. These results provide a basis for screening the chitinase-based biological pesticide.
Animals ; Bombyx ; enzymology ; Chitinases ; isolation & purification ; metabolism ; Enzyme Stability ; Insect Proteins ; isolation & purification ; metabolism ; Temperature

OBJECTIVE: To evaluate the value of chitinase-like protein YKL-40 in bronchoalveolar lavage fluid (BALF) for predicting refractory METHODS: A total of 50 children with common RESULTS: Compared with the common MPP group, the RMPP group had significantly higher incidence rates of fever, shortness of breath, lung consolidation, and pleural effusion ( CONCLUSIONS There is an increase in the level of YKL-40 in BALF in children with RMPP, and the level of YKL-40 in BALF has a certain value for predicting RMPP.
Bronchoalveolar Lavage Fluid ; Child ; Chitinase-3-Like Protein 1 ; Chitinases ; Humans ; Mycoplasma pneumoniae ; Pneumonia, Mycoplasma/diagnosis*

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.
Acetylglucosamine ; Acetylglucosaminidase ; Bacillus coagulans ; Chitin ; Chitinases ; Hydrogen-Ion Concentration ; Recombinant Proteins/genetics*

Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.
Biocatalysis ; Chitin ; analogs & derivatives ; Chitinases ; Hydrogen-Ion Concentration ; Polymerase Chain Reaction

To improve the expression of heterologous genes using baculovirus expression system, we constructed a novel shuttle vector based on the Bm-Bacmid. In the Bm-Bacmid, partial sequences of Chitinase and Cystein Protease were replaced with a tandem cassette of Cm and egfp through homologous recombination. Bombyx mori bidensovirus (BmBDV) ns1 under the control of polyhedrin promoter was inserted into the modified Bm-bacmid by transposition. For comparison, BmBDV ns1 under the control of polyhedrin promoter was also cloned in the wild type Bm-bacmid. The resulting Bm-bacmids were transfected into the cultured BmN cells to prepare recombinant virus to infect silkworms for expression of BmBDV ns1. Total proteins of hemocyte from infected silkworms were subjected to Western blotting and ELISA analysis. The yield of BmBDV NS1 1 with the modified vector was three times as much as that with the unmodified vector. The method to improve the yield of BmBDV NS1 in silkworms will facilitate the function and three-dimensional structure study of BmBDV NS1.
Animals ; Baculoviridae ; Bombyx ; virology ; Cell Line ; Chitinases ; Cysteine Proteases ; Genetic Vectors ; Insect Viruses ; Promoter Regions, Genetic ; Transfection

The principal component analysis (PCA) was applied to the data processing in training sets, the new principal components were then used as input data for support vector machine model. A prediction model for optimum pH of chitinase was established based on uniform design. When The regularized constant C, epsilon and Gamma were 10, 0.7 and 0.5 respectively, the calculated pHs fitted the reported optimum pHs of chitinase very well and the MAPEs (Mean Absolute Percent Error) was 3.76%. At the same time, the predicted pHs fitted the reported optimum pHs well and the MAE (Mean Absolute Error) was 0.42 pH unit. It was superior in fittings and predictions compared to the model based on back propagation (BP) neural network.
Algorithms ; Animals ; Chitinases ; chemistry ; metabolism ; Humans ; Hydrogen-Ion Concentration ; Models, Biological ; Models, Statistical ; Neural Networks (Computer) ; Principal Component Analysis

OBJECTIVE: To examine AMCase mRNA expression levels in nasal mucosa of allergic rhinitis SD rats. METHOD: Thirty SD rats were chosen and randomly divided into the experimental group and the control group. 20 in experimental group and 10 in the control group. AR model rats were established through repeated intraperitoneal shot of ovalbumin (OVA) for 2 weeks and consequently confirmed by local challenge with OVA for 1 week. The control group was treated by the same method with Physiological saline water instead of OVA. After the last excitation allergic rhinitis was diagnosed according to the accumulation score about nasal symptom. The septal mucosa of all rats were used to diagnose pathologically by HE dyeing. AMCase mRNA in nasal mucosa, obtained from the bilateral nasal mucosa in two groups, were used to do reverse transcriptive polymerase chain reaction. Real-time polymerase chain reaction was used to examine AMCase mRNA expression levels. RESULT: (1) The results showed definitely that there were positive expression of AMCase mRNA in normal nasal mucosa. This expression increase significantly during nasal allergy (P < 0.05). (2) The increased expression of AMCase mRNA in allergic rhinitis are related with the nasal symptoms score (r = 0.411, P < 0.05). CONCLUSION Increased expression of AMCase mRNA in nasal mucosa in allergic rhinitis model might play roles in the pathogenesis of AR, and Restrain the enzyme activity could become new treatment targets of allergic rhinitis.
Animals ; Chitinases ; metabolism ; Nasal Mucosa ; metabolism ; Ovalbumin ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Rhinitis, Allergic ; enzymology ; metabolism

Chitinases were produced by a lot of microorganisms. Chitinase gene expression in most of the chitinase producing bacteria was inducible by chitin. Low levels of chitinase were observed in the presence of glucose. To date, however, the regulation of such chitinase gene in Bacillus thuringiensis had not been well studied. In this paper, all 77 Bacillus thuringiensis strains were grown in the medium with or without chitin. We measured quantitatively the chitinase activity of the cultures. Moreover, we investigated the suppressive effect of glucose on chitinase of 4 strains. Also we studied the relationship between chitin induction and glucose suppression on chitinase. This investigation demonstrated that all tested B. thuringiensis strains could produce chitinase without chitin. After induction, the chitinolytic activity of 31 tested strains had no obvious response to the inducer, whereas 44 stains increased in different degree. Among these strains, most of them did not markedly increase the levels of chitinase, and many stains simultaneously displayed the expression mode of inducible and constitutive. The glucose inhibited the inductive effect of chitin, but it could not inhibit the basal expression of chitinase. Two strains No. 38 and No. 75 belonged to different expression types. But we just found several different bases in the regulatory region of chitinase genes chiA and chiB from them.
Bacillus thuringiensis ; enzymology ; growth & development ; Base Sequence ; Chitin ; pharmacology ; Chitinases ; biosynthesis ; genetics ; Culture Media ; chemistry ; Culture Techniques ; Glucose ; pharmacology ; Molecular Sequence Data ; Polymorphism, Genetic