活性口袋关键色氨酸对几丁质酶Chi304催化性质的研究

doi:10.13304/j.nykjdb.2021.0877

摘要/Abstract

摘要：

几丁质酶中存在结合口袋及水解活性位点，关键氨基酸与底物几丁质的结合可调控酶的水解活性。以嗜热几丁质酶Chi304为研究材料，采用同源建模分析其高级结构，发现在其底物结合口袋附近存在2个色氨酸（W140与W272），将其定点突变为丙氨酸后，采用高效液相色谱检测酶的水解产物，并进一步分析水解产物三乙酰壳三糖（DP₃）与二乙酰壳二糖（DP₂）的比例（DP₃/DP₂），用于评估突变体的效果。结果表明，突变体W140A、W272A与W140/272A水解胶体几丁质产物DP₃/DP₂分别较野生型提升23.3%、45.7%与80.0%。由此表明，W140与W272是影响酶与底物结合的关键氨基酸，将其突变为丙氨酸可提升Chi304的内切活力，降低外切活力。

关键词: 几丁质酶, 定点突变, 几丁寡糖, 理性设计

Abstract:

There are binding sites and hydrolytic active sites in chitinase， and the binding of key amino acids to substrate chitin can appropriately regulate the hydrolytic activity of the enzyme. The thermophilic chitinase Chi304 was as experimental material. Swiss-Model and analysis of its advanced structure revealed the presence of two tryptophans （W140 and W272） near its substrate binding pocket. And these two tryptophans were site-directed mutated to alanine. High performance liquid chromatography was used to detect the hydrolysis products of the enzyme. The ratio of product triacetyl chitosaccharide to diacetyl chitosaccharide （DP₃/DP₂） was used to evaluate the effect of the mutants. The mutants （W140A， W272A and W140/272A） hydrolyzed colloidal chitin， and the proportions of product （DP₃/DP₂） were increased by 23.3%， 45.7% and 80.0% compared with that of wild type， respectively. The results showed that W140 and W272 were the key amino acids affecting the binding of enzyme to substrate， and the mutation of alanine to Chi304 increased the endogenous activity and decreased the exogenous activity.

Key words: chitinase, site-directed mutation, chitin oligosaccharide, rational design

中图分类号:

Q556+.2

田晓倩, 卢海强, 伍宁丰, 田健, 关菲菲. 活性口袋关键色氨酸对几丁质酶Chi304催化性质的研究[J]. 中国农业科技导报, 2023, 25(2): 76-82.

Xiaoqian TIAN, Haiqiang LU, Ningfeng WU, Jian TIAN, Feifei GUAN. Catalytic Properties of the Active Pocket Key Tryptophan on Chitinase Chi304[J]. Journal of Agricultural Science and Technology, 2023, 25(2): 76-82.

图/表 5

图1 Chi304结构信息A：Chi304蛋白三维结构，红色为α-螺旋，蓝色为β-折叠，黑色label为活性位点；B：Chi304蛋白结构surface展示，灰色为整体结构，红色为深沟表面色氨酸，黑色label为两个色氨酸位点

Fig. 1 Structure of Chi304A： Protein structure diagram of Chi304； red indicateds α -helix， blue indicateds β-fold， black labels are the active sites； B： Chi304 protein structure surface display diagram； gray indicateds the overall structure， red indicateds deep groove surface tryptophans， black labels are the two tryptophan sites

图2 Chi304突变体SDS-PAGE分析

Fig. 2 SDS-PAGE analysis of Chi304 mutants

图3 Chi304及突变体的水解产物注：****表示与Chi304间差异在P<0.000 1水平显著。

Fig. 3 Hydrolysate of Chi304 and mutantsNote：**** indicates significante differences with Chi304 at P<0.000 1 level.

图4 W140A/W272A的SDS-PAGE分析

Fig. 4 SDS-PAGE analysis of W140A/W272A

图5 Chi304与W140A/W272A水解（GlcNAc）6分析A：Chi304水解（GlcNAc）6时程HPLC分析；B：W140A/W272A水解（GlcNAc）6时程HPLC分析

Fig.5 Analysis of hydrolyzed （GlcNAc）6 by Chi304 and W140A/W272AA： HPLC analysis of Chi304 hydrolyzed （GlcNAc）6； B： HPLC analysis of W140A/W272A hydrolyzed （GlcNAc）6

参考文献 34

1	KURITA K. Controlled functionalization of the polysaccharide chitin [J]. Prog. Polym. Sci., 2001, 26(9):1921-1971.
2	CRINI G. Historical review on chitin and chitosan biopolymers [J]. Environ. Chem. Lett., 2019, 17(4):1623-1643.
3	PARK B K, KIM M M. Applications of chitin and its derivatives in biological medicine [J]. Int. J. Mol. Sci., 2010, 11(12):5153-5165.
4	WANG J, ZHANG J Q, SONG F G, et al.. Purification and characterization of chitinases from ridgetail white prawn exopalaemon carinicauda [J]. Molecules, 2015, 20(2):1955-1967.
5	RAHMAN M H, HJELJORD L G, AAM B B, et al.. Antifungal effect of chito-oligosaccharides with different degrees of polymerization [J]. Eur. J. Plant Pathol., 2015, 141(1):147-158.
6	LUO C, LIU W J, LUO B H, et al.. Antibacterial activity and cytocompatibility of chitooligosaccharide-modified polyurethane membrane via polydopamine adhesive layer [J]. Carbohydr. Polym., 2017, 156:235-243.
7	TSUKADA K, MATSUMOTO T, AIZAWA K, et al.. Antimetastatic and growth-inhibitory effects of N-acetylchitohexaose in mice bearing Lewis lung carcinoma [J]. Jap. J. Cancer Res., 1990, 81(3):259-265.
8	VO T S, NGO D H, TA Q V, et al.. Protective effect of chitin oligosaccharides against lipopolysaccharide-induced inflammatory response in BV-2 microglia [J]. Cell Immunol., 2012, 277(1):14-21.
9	LOMBARD V, GOLACONDA RAMULU H, DRULA E, et al.. The carbohydrate-active enzymes database (CAZy) in 2013 [J]. Nucleic Acids Res., 2014, 42(D1):490-495.
10	FUNKHOUSER J D, ARONSON N N. Chitinase family GH18:evolutionary insights from the genomic history of a diverse protein family [J]. BMC Evol. Biol., 2007, 7(1):96-112.
11	GOODAY G W. Aggressive and defensive roles for chitinases [J]. EXS, 1999, 87:157-169.
12	CHOU Y T, YAO S, CZERWINSKI R, et al.. Kinetic characterization of recombinant human acidic mammalian chitinase [J]. Biochemistry, 2006, 45(14):4444-4454.
13	SUZUKI K, TAIYOJI M, SUGAWARA N, et al.. The third chitinase gene (chiC) of Serratia marcescens 2170 and the relationship of its product to other bacterial chitinases [J]. Biochem. J., 1999, 343(3):587-596.
14	PANTOOM S, VETTER I R, PRINZ H, et al.. Potent family-18 chitinase inhibitors:x-ray structures, affinities, and binding mechanisms [J]. J. Biol. Chem., 2011, 286(27):24312-24323.
15	LI H, GREENE L H. Sequence and structural analysis of the chitinase insertion domain reveals two conserved motifs involved in chitin-binding [J/OL]. PLoS ONE, 2010, 5(1):e8654 [2021-09-10]. .
16	PAYNE C M, BABAN J, HORN S J, et al.. Hallmarks of processivity in glycoside hydrolases from crystallographic and computational studies of the Serratia marcescens chitinases [J]. J. Biol. Chem., 2012, 287(43):36322-36330.
17	MALECKI P H, RACZYNSKA J E, VORGIAS C E, et al.. Structure of a complete four-domain chitinase from Moritella marina, a marine psychrophilic bacterium [J]. Acta Cryst., 2013, 69(5):821-829.
18	FUNKHOUSER J D, ARONSON N N. Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family [J/OL]. BMC Evol. Biol., 2007, 7:96 [2021-09-10]. .
19	WU Q, LIU T, YANG Q. Cloning, expression and biocharacterization of OfCht 5, the chitinase from the insect Ostrinia furnacalis [J]. Insect Sci., 2013, 20(2):147-157.
20	KROLICKA M, HINZ S W A, KOETSIER M J, et al.. Chitinase Chi1 from Myceliophthora thermophila C1, a thermostable enzyme for chitin and chitosan depolymerization [J]. J. Agric. Food Chem., 2018, 66(7):1658-1669.
21	关菲菲,伍宁丰,田健,等.嗜热几丁质酶Chi 304突变体及其在降解几丁质中的应用[P].中国, CN112899257A.
22	ROBERTS W K, SELITRENNIKOFF C P. Plant and bacterial chitinases differ in antifungal activity [J]. Microbiology, 1988, 134(1):169-176.
23	PREETY, SHARMA S, HOODA V. Purification and analytical application of Vigna mungo chitinase for determination of total fungal load of stored cereals [J]. Appl. Biochem. Biotechnol., 2018, 186(1):12-26.
24	OYELEYE A, NORMI Y M. Chitinase:diversity, limitations, and trends in engineering for suitable applications [J/OL]. Biosci. Rep., 2018, 38(4):BSR20180323 [2021-09-10]. .
25	OHNUMA T, NUMATA T, OSAWA T, et al.. Crystal structure and mode of action of a class V chitinase from Nicotiana tabacum [J]. Plant Mol. Biol., 2011, 75(3):291-304.
26	SIKORSKI P, SØRBOTTEN A, HORN S J, et al.. Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan [J]. Biochemistry, 2006, 45(31):9566-9574.
27	HORN S J, SORBOTTEN A, SYNSTAD B, et al.. Endo/exo mechanism and processivity of family 18 chitinases produced by Serratia marcescens [J]. FEBS J., 2006, 273(3):491-503.
28	LIU T, HAN H Y, WANG D, et al.. Potent fungal chitinase for the bioconversion of mycelial waste [J]. J. Agric. Food Chem., 2020, 68(19):5384-5390.
29	JANA S, HAMRE A G, WILDBERGER P, et al.. Aromatic-mediated carbohydrate recognition in processive Serratia marcescens chitinases [J]. JPCB, 2016, 120(7):1236-1249.
30	HAMRE A G, JANA S, REPPERT N K, et al.. Processivity, substrate positioning, and binding: the role of polar residues in a family 18 glycoside hydrolase [J]. Biochemistry, 2015, 54(49):7292-7306.
31	WU S, WU S. Processivity and the mechanisms of processive endoglucanases [J]. Appl. Biochem. Biotechnol., 2020, 190(2):448-463.
32	CHUNDAWAT S P S, NEMMARU B, HACKL M, et al.. Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose Ⅲ [J]. J. Biol. Chem., 2021, 296:100431-100448.
33	VARUN T K, SENANI S, KUMAR N, et al.. Extraction and characterization of chitin, chitosan and chitooligosaccharides from crab shell waste [J]. Indian J. Anim. Res., 2017, 51(6):1066-1072.
34	SYNSTAD B, OSLAS H, RNA R, et al.. Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method [J]. Biosci. Biotechnol. Biochem., 2008, 72(3):715-723.

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