Abstract：Chitinases (CHI) are a series of glycoside hydrolases (GHs) that break chitin into chitooligosaccharides ((GlcNAc)n, 10≥n≥2) or N-acetylglucosamine (GlcNAc). In this study, a chitinase gene derived from Bacillus thuringensis, BtCHI1, was cloned and heterologously expressed in Escherichia coli BL21(DE3)plysS. The enzymatic properties, kinetic parameters, and substrate hydrolytic patterns recombinant enzyme BtCHI1 were characterized. The results showed that the molecular mass of BtCHI1 was approximate 75 kD. Its optimum temperature and pH were 40 ℃ and 7.0, respectively. The enzyme was unstable after heat-challenge at 50 ℃ for 1 h, retaining only 20.11% residual activity. However, the enzyme was considerably stable in broad pH buffers ranging from 3.0 to 10.0, retained over 80% residual activities after incubation for 1 h, respectively. Substrate kinetics assays indicated the recombinant BtCHI1 was active against chitin, colloidal chitin and chitosan, with Vmax values of (4.62±0.46), (0.52±0.03) and (0.22±0.02) μmol/(min·mg), respectively. Nevertheless, the enzyme was inactive towards hydroxypropyl chitosan or glycol chitosan. Meanwhile, the enzyme was considerably resilient to 0.5~10 mmol/L Na+, Al3+ or EDTA (P>0.05). However, after treatment with 0.5~10 mmol/L Ca2+ or 5~10 mmol/L SDS for 1 h, the activities of BtCHI1 decline significantly (P<0.01). Thin-layer chromatography and high-performance liquid chromatography analysis revealed that BtCHI1 releases chitobiose from chitin and colloidal chitin, and further converted it into GlcNAc. Additionally, BtCHI1 (0.28 U) was observed to liberate (1.00±0.04) mg/mL and (1.71±0.11) mg/mL from natural shrimp and crab shell substrates. These results provide new insights into chitooligosaccharides/GlcNAc development and natural chitin substrate utilization.
[1] 陈立功, 张庆芳, 迟乃玉, 等. 2020. 低温几丁质酶基因在乳酸克鲁维酵母中的重组表达及酶学性质表征[J]. 微生物学报, 60(1): 172-182. (Chen L G, Zhang Q F, Chi N Y, et al.2020. Recombinant expression and characterization of cold-adapted chitinase genes in Kluyveromyces lactis[J]. Acta Microbiologica Sinica, 60(1): 172-182.) [2] 冯俊丽, 朱旭芬. 2004. 微生物几丁质酶的分子生物学研究[J]. 浙江大学学报(农业与生命科学版), 30(1): 102-108. (Feng J L, Zhu X F.2004. Molecular biology of chitinase in microbe[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 30(1): 102-108.) [3] 冯玉和, 孙小宝, 陈书昕, 等. 2020. 米曲霉裂解性多糖单加氧酶的异源表达与性质分析[J]. 微生物学报, 60(01): 183-199. (Feng Y H, Sun X B, Chen S X, et al.2020. Heterologous expression and characterization of Aspergillus oryzae lytic polysaccharide monooxygenases[J]. Acta Microbiologica Sinica, 60(1): 183-199.) [4] 巩凯玲, 陈双慧, 纪晓晨, 等. 2019. 植物几丁质酶的研究进展[J]. 分子植物育种, 17(20): 6840-6849. (Gong K L, Chen S H, Ji X C, et al.2019. The research progress of plant chitinase[J]. Molecular Plant Breeding, 17(20): 6840-6849.) [5] 谷天燕, 刘晓楠, 李玲聪, 等. 2019. 两端融合表达几丁质结合结构域提高几丁质酶抗真菌活性[J]. 微生物学报, 59(4): 762-770. (Gu T Y, Liu X N, Li L C, et al.2019. Enhancing antifungal activity of chitinase by fusion of chitin binding domain at both termini[J]. Acta Microbiologica Sinica, 59(4): 762-770.) [6] 何迎春, 李小湘, 高必达. 2003. 含粘质沙雷氏菌几丁质酶SchiA基因的植物转化质粒pBG1112构建和水稻遗传转化[J].农业生物技术学报, 11(2): 121-126. (He Y C, Li X X, Gao B D.2003. Construction of a new plant transformation vector pBG1112 with a chitinase gene SchiA from Serratia marcescens and its genetic transformation in rice[J]. Chinese Journal of Agricultural Biotechnology, 11(2): 121-126.) [7] 姜红鹰, 周玉玲, 张桂敏, 等. 2016. 酶法制备甲壳素系列衍生物的研究进展[J]. 氨基酸和生物资源, 38(4): 71-76. (Jiang H Y, Zhou Y L, Zhang G M, et al.2016. Research progress on production of chitin and its derivatives through enzymatic hydrolysis[J]. Amino Acid & Biotic Resources, 38(4): 71-76.) [8] 姜竹峪, 陈羽, 魏锦兴, 等. 2016. 几丁质酶的研究进展[J]. 沈阳药科大学学报, 33(5): 414-418. (Jiang Z Y, Chen Y, Wei J X, et al.2016. Research advances in research on chitinase[J]. Journal of Shenyang Pharmaceutical University, 33(5): 414-418.) [9] 刘东. 2010. 苏云金芽胞杆菌几丁质酶特性分析及其基因启动子序列的确定[D]. 博士学位论文, 南开大学, 导师: 陈月华, pp. 35-36. (Liu D.2010. Characterizations of chitinases from Bacillus thuringiensis subsp colmeri 15A3 and identification of promoter sequence of chiB[D]. Dissertation for PhD., Nankai University, Supervisor: Chen YH, pp. 35-36.) [10] 潘禺. 2020. 微囊藻毒素降解酶MlrA的酶学特性表征及其结构功能解析[D].硕士学位论文, 江西理工大学, 导师: 王华生, pp. 56-57. (Pan Y.2020. Biochemical characterisation and structure-function analysis of MlrA fron microcystin-derading bacteria[D]. Thesis for M.S., Jiangxi University of Science and Technology, Supervisor: Wang H S, pp. 56-57.) [11] 周可鑫, 王欢, 朱鑫涛, 等. 2020. 利用分子环化技术提高瘤胃微生物木聚糖酶热稳定性[J]. 生物工程学报, 36(5): 920-931. (Zhou K X, Wang H, Zhu X T, et al.2020. Enhancing thermostability of xylanase from rumen microbiota by molecular cyclization[J]. Chinese Journal of Biotechnology, 36(5): 920-931.) [12] Arnold N D, Brück W M, Garbe D, et al.2020. Enzymatic modification of native chitin and conversion to specialty chemical products[J]. Marine Drugs, 18(2): 93. [13] Beier S, Bertilsson S.2013. Bacterial chitin degradation-mechanisms and ecophysiological strategies[J]. Frontiers in Microbiology, 4: 149. [14] Beygmoradi A, Homaei A, Hemmati R, et al.2018. Marine chitinolytic enzymes, a biotechnology treasure hidden in the ocean?[J]. Applied Microbiology and Biotechnology, 102(23): 9937-9948. [15] Bradford M M.1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 72(1/2): 248-254. [16] Charley C S, Livia K, Irina L, et al.2013. Metarhizium anisopliae chitinase CHIT30 is involved in heat-shock stress and contributes to virulence against Dysdercus peruvianus. Fungal Biology[J]. 117(2): 137-144. [17] Chen J K, Shen C R, Liu C L.2010. N-acetylglucosamine: Production and applications[J]. Marine Drugs, 8(9): 2493-2516. [18] Estela C M,Tina R T,Gustav V K, et al.2018. Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions[J]. Journal of Biological Chemistry, 293(10): 3849-3859. [19] Gao DY, Sun X B, Liu M Q, et al.2019. Characterization of thermostable and chimeric enzymes via isopeptide bond-mediated molecular cyclization[J]. Journal of Agricultural and Food Chemistry, 37: 6837-6846. [20] Gao X, Chen X, Zhang J, et al.2016. Transformation of chitin and waste shrimp shells into acetic acid and pyrrole. ACS Sustainable Chemistry and Engineering, 4(7): 3912-3920. [21] Gasmi M, Kitouni M, Carro L, et al.2019. Chitinolytic actinobacteria isolated from an Algerian semi-arid soil: development of an antifungal chitinase-dependent assay and GH18 chitinase gene identification[J]. Annals of Microbiology, 69(4): 395-405. [22] Grégorio C.2019. Historical review on chitin and chitosan biopolymers[J]. Environmental Chemistry Letters, 17(1): 1623-1642. [23] Guo X X, Xu P, Zong M H, et al.2017. Purification and characterization of alkaline chitinase from Paenibacillus pasadenensis CS0611[J]. Chinese Journal of Catalysis, 38(4): 665-672. [24] Hegazy U M, El-Khonezy M I, Shokeer A, et al.2019. Revealing of a novel xylose-binding site of Geobacillus stearothermophilus xylanase by directed evolution[J]. Journal of Biochemistry, 165(2): 177-184. [25] He J, Tang F, Chen D, et al.2019. Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei[J]. PLOS ONE, 14(1): e0210548. [26] Huo F, Ran C, Yang Y, et al.2016. Gene cloning, expression and characterization of an exo-chitinase with high β-glucanase activity from Aeromonas veronii B565[J]. Acta Microbiologiga Sinica, 56(5): 787-803. [27] Khan F I, Rahman S, Queen A, et al.2017. Implications of molecular diversity of chitin and its derivatives[J]. Applied Microbiology and Biotechnology, 101(9): 3513-3536. [28] Krolicka M, Hinz S WA, Koetsier M J, et al.2018. Chitinase Chi1 from Myceliophthora thermophila C1, a thermostable enzyme for chitin and chitosan depolymerization[J]. Journal of Agricultural and Food Chemistry, 66(7): 1658-1669. [29] Langner T, Ö?ztÖ?ü?rk M, Hartmann S, et al.2015. Chitinases are essential for cell separation in Ustilago maydis[J]. Eukaryotic Cell, 14(9): 846-857. [30] Lombard V, Golaconda R H, Drula E, et al.2014. The carbohydrate-active enzymes database (CAZy) in 2013[J]. Nucleic Acids Research, 42(D1): D490-D495. [31] Miller GL.1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical Biochemistry, 31(3): 426-428. [32] Nakatani K, Katano Y, Kojima K, et al.2018. Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library[J]. Bioscience Biotechnology and Biochemistry, 82(10): 1715-1723. [33] Rita S, Kirti S, Antaryami S.2017. Chitin and chitosan: biopolymers for wound management[J]. International Wound Journal, 14(6): 1276-1289. [34] Rosas-García N M, Fortuna-González JM, Barboza-Corona JE.2013. Characterization of the chitinase gene in Bacillus thuringiensis Mexican isolates[J]. Folia Microbiologica, 58(6): 483-490. [35] Sun X B, Cao J W, Wang J K, et al.2019. SpyTag/SpyCatcher molecular cyclization confers protein stability and resilience to aggregation[J]. New Biotechnology, 49: 28-36. [36] Wang Y J, Jiang W X, Zhang Y S, et al.2019. Structural insight into chitin degradation and thermostability of a novel endochitinase from the glycoside hydrolase family 18[J]. Frontiers in Microbiology, 10: 2457. [37] Wennmann J T, Alleti G G, Jehle J A.2015. The genome sequence of Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) reveals a new baculovirus species within the Agrotis baculovirus complex[J]. Virus Genes, 50(2): 260-276. [38] Xu J, Yang Y L, Liu Y, et al.2016.Characterization of an exo-chitinase from a Citrobacter strain isolated from the intestine content of large yellow croakers[J]. Acta Microbiologica Sinica, 56(7): 1089-1104. [39] Yang S Q, Fu X, Yan Q J, et al.2016. Cloning, expression, purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii[J]. Food Chemistry, 192: 1041-1048. [40] Zhang A, He Y M, Wei G G, et al.2018. Molecular characterization of a novel chitinase CmChi1 from Chitinolyticbacter meiyuanensis SYBC-H1 and its use in N-acetyl-D-glucosamine production[J]. Biotechnology for Biofuels, 11: 179. [41] Zhang Y, An R, Yatsunami R, et al.2010. Characterization of a haloarchaeal GH family 18 chitinase with additional acidic amino acids on its protein surface[J]. Journal of Japanese Society for Extremophiles, 9(2): 72-74. [42] Zhou Y J, Kang L Q, Niu X, et al.2016. Purification, characterization and physiological significance of a chitinase from the pilei of Coprinopsis cinerea fruiting bodies[J]. FEMS Microbiology Letters, 363(12): fnw120.
