Identification of key amino acid sites for pH  stability of GH46 family chitosanase

CHENG Yimeng; SUN Huihui; LIU Qi; ZHAO Ling; CAO Rong

doi:10.12131/20210290

Chitooligosaccharides, which have a variety of biological activities, are the only known basic oligosaccharide widely used in food, agriculture and biomedicine. Chitosanases can cleave the β-1,4 glycosidic bonds in chitosan specifically to form chitooligosaccharides with different degrees of polymerization. Therefore, obtaining chitosanases with good stability is the key for the large-scale enzymatic preparation of chitooligosaccharides. In order to identify the amino acid sites affecting the pH stability of GH46 family chitosanases, the chitosanase from Bacillus sp. DAU101 (optimal pH 7.5) was selected as template and the chitosanase Csn-BAC from Bacillus sp. MD-5 as the research object. By combining homology modeling and sequence alignments, four candidate sites were selected, and the corresponding mutants were obtained (V1: P68A; V2: A137G; V3: A203M; V4: H234E). Compared with Csn-BAC, the thermal stabilities of four mutants showed varying degrees of reduction, while the pH stability was significantly improved. These results indicate that the selected amino acid sites have an obvious effect on pH tolerance, and this strategy is an effective way to modify the stability of chitosanase.

[1]	BHUVANACHANDRA B, SIVARAMAKRISHNA D, ALIM S, et al. New class of chitosanase from Bacillus amyloliquefaciens for the generation of chitooligosaccharides[J]. J Agr Food Chem, 2021, 69(1): 78-87. doi: 10.1021/acs.jafc.0c05078
[2]	YANG G, SUN H, CAO R, et al. Characterization of a novel glycoside hydrolase family 46 chitosanase, Csn-BAC, from Bacillus sp. MD-5[J]. Int J Biol Macromol, 2020, 146: 518-523. doi: 10.1016/j.ijbiomac.2020.01.031
[3]	吴玉潇, 徐海涛, 高云华, 等. 壳寡糖的生物活性研究进展[J]. 明胶科学与技术, 2015, 35(3): 128-132. doi: 10.3969/j.issn.1004-9657.2015.03.004
[4]	ZOU P, YANG X, WANG J, et al. Advances in characterisation and biological activities of chitosan and chitosan oligosaccharides[J]. Food Chem, 2016, 190: 1174-1181. doi: 10.1016/j.foodchem.2015.06.076
[5]	YANG F, LUAN B, SUN Z, et al. Application of chitooligosaccharides as antioxidants in beer to improve the flavour stability by protecting against beer staling during storage[J]. Biotechnol Lett, 2017, 39(2): 305-310. doi: 10.1007/s10529-016-2248-3
[6]	MUANPRASAT C, CHATSUDTHIPONG V. Chitosan oligosaccharide: biological activities and potential therapeutic applications[J]. Pharmacol Ther, 2016, 170: 80-97.
[7]	潘珍, 程冬冬, 位晓娟, 等. 壳五糖对骨肉瘤细胞抗肿瘤作用的研究[J]. 国际骨科学杂志, 2020, 41(2): 114-120. doi: 10.3969/j.issn.1673-7083.2020.02.011
[8]	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: 147-158. doi: 10.1007/s10658-014-0533-3
[9]	MEI Y X, DAI X Y, YANG W, et al. Antifungal activity of chitooligosaccharides against the dermatophyte Trichophyton rubrum[J]. Int J Biol Macromol, 2015, 77: 330-335.
[10]	DAS S N, MADHUPRAKASH J, SARMA P V S R N, et al. Biotechnological approaches for field applications of chitooligosaccharides (COS) to induce innate immunity in plants[J]. Crit Rev Biotechnol, 2015, 35(1): 29-43. doi: 10.3109/07388551.2013.798255
[11]	KRITCHENKOV A S, KLETSKOV A V, EGOROV A R, et al. New water-soluble chitin derivative with high antibacterial properties for potential application in active food coatings[J]. Food Chem, 2021, 343: 128696. doi: 10.1016/j.foodchem.2020.128696
[12]	LIAQAT F, ELTEM R. Chitooligosaccharides and their biological activities: a comprehensive review[J]. Carbohydr Polym, 2018, 184: 243-259. doi: 10.1016/j.carbpol.2017.12.067
[13]	鲁晶娣. Bacillus nakamurai壳聚糖酶的酶学性质及其活性位点研究[D]. 柳州: 广西科技大学, 2019: 1-2.
[14]	李燕. 壳聚糖酶解及降解产物的分析与应用[D]. 天津: 天津科技大学, 2018: 1-4.
[15]	THADATHIL N, VELAPPAN S P. Recent developments in chitosanase research and its biotechnological applications: a review[J]. Food Chem, 2014, 150: 392-399. doi: 10.1016/j.foodchem.2013.10.083
[16]	SUN H, GAO L, XUE C, et al. Marine-polysaccharide degrading enzymes: status and prospects[J]. Compr Rev Food Sci F, 2020, 19: 2767-2796. doi: 10.1111/1541-4337.12630
[17]	SUN H, MAO X, GUO N, et al. Discovery and characterization of a novel chitosanase from Paenibacillus dendritiformis by phylogeny-based enzymatic product specificity prediction[J]. J Agr Food Chem, 2018, 66: 4645-4651. doi: 10.1021/acs.jafc.7b06067
[18]	SUN H, YANG G, CAO R, et al. Expression and characterization of a novel glycoside hydrolase family 46 chitosanase identified from marine mud metagenome[J]. Int J Biol Macromol, 2020, 159: 904-910. doi: 10.1016/j.ijbiomac.2020.05.147
[19]	QIN Z, CHEN Q M, LIN S, et al. Expression and characterization of a novel cold-adapted chitosanase suitable for chitooligosaccharides controllable preparation[J]. Food Chem, 2018, 253(1): 139-147.
[20]	GUO N, SUN J, WANG W, et al. Cloning, expression and characterization of a novel chitosanase from Streptomyces albolongus ATCC 27414[J]. Food Chem, 2019, 286: 696-702. doi: 10.1016/j.foodchem.2019.02.056
[21]	QU G, ZHAO J, ZHENG P, et al. Recent advances in directed evolution[J]. Chin J Biotech, 2018, 34(1): 1-11.
[22]	YANY Y, ZHENG Z, XIAO Y et al. Cloning and characterization of a cold-adapted chitosanase from marine bacterium Bacillus sp. BY01[J]. Molecules, 2019, 24(21): 3915. doi: 10.3390/molecules24213915
[23]	WANG Y, QIN Z, FAN L, et al. Structure-function analysis of Gynuella sunshinyii chitosanase uncovers the mechanism of substrate binding in GH family 46 members[J]. Int J Biol Macromol, 2020, 165: 2038-2048. doi: 10.1016/j.ijbiomac.2020.10.066
[24]	LIU Y, LI Y, TONG S, et al. Expression of a Beauveria bassiana chitosanase (BbCSN-1) in Pichia pastoris and enzymatic analysis of the recombinant protein[J]. Protein Expres Purif, 2019, 166: 105519.
[25]	YOON H, KIM H, LIM Y, et al. Thermostable chitosanase from Bacillus sp. strain CK4: cloning and expression of the gene and characterization of the enzyme[J]. Appl Environ Microb, 2000, 66(9): 3727-3734. doi: 10.1128/AEM.66.9.3727-3734.2000
[26]	LEE Y S, YOO J S, CHUNG S Y, et al. Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101[J]. Appl Microbiol Biot, 2006, 73(1): 113-121. doi: 10.1007/s00253-006-0444-0
[27]	LARKIN M A, BLACKSHIELDS G, BROWN N P, et al. Clustal W and Clustal X version 2.0[J]. Bioinformatics, 2007, 23(21): 2947-2948. doi: 10.1093/bioinformatics/btm404
[28]	BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72(1/2): 248-254.
[29]	LI Y, GOU Y, LIU Z, et al. Structure-based rational design of chitosanase CsnMY002 for high yields of chitobiose[J]. Colloid surfaces B, 2021, 202(8): 111692.
[30]	ZHANG J, CAO H, LI S, et al. Characterization of a new family 75 chitosanase from Aspergillus sp. W-2[J]. Int J Biol Macromol, 2015, 81: 362-369. doi: 10.1016/j.ijbiomac.2015.08.026
[31]	王琦, 崔阳, 刘进宝, 等. 壳聚糖酶的基因克隆表达及酶学性质研究[J]. 食品与生物技术学报, 2019, 38(1): 147-155. doi: 10.3969/j.issn.1673-1689.2019.01.022
[32]	王亚妮. GH46家族壳聚糖酶的结构解析与催化机理研究[D]. 上海: 华东理工大学, 2020: 14.
[33]	杨光, 盛军, 陈亚东, 等. 枯草芽孢杆菌壳聚糖酶基因在毕赤酵母中的表达及酶学性质[J]. 中国水产科学, 2017, 24(6): 1288-1297.
[34]	XING P, LIU D, YU W G, et al. Molecular characterization of an endo-type chitosanase from the fish pathogen Renibacterium sp. QD1[J]. J Mar Biol Assoc UK, 2014, 94(4): 681-686. doi: 10.1017/S0025315413001859
[35]	罗洒. 新型壳聚糖酶的高效表达及壳寡糖制备工艺研究[D]. 上海: 华东理工大学, 2019: 21.
[36]	GUPTA V, PRASANNA R, SRIVASTAVA A K, et al. Purification and characterization of a novel antifungal endo-type chitosanase from Anabaena fertilissima[J]. Ann Microbiol, 2012, 62(3): 1089-1098. doi: 10.1007/s13213-011-0350-2
[37]	CHEN X, ZHAi C, KANG L, et al. High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris[J]. Biotechnol Lett, 2012, 34(4): 689-694. doi: 10.1007/s10529-011-0816-0
[38]	ZHOU Y, CHEN X, LI X, et al. Purification and characterization of a new cold-adapted and thermo-tolerant chitosanase from marine bacterium Pseudoalteromonas sp. SY39[J]. Molecules, 2019, 24(1): 183. doi: 10.3390/molecules24010183
[39]	MA C, LI X, YANG K, et al. Characterization of a new chitosanase from a marine Bacillus sp. and the anti-oxidant activity of its hydrolysate[J]. Mar Drugs, 2020, 18(2): 126. doi: 10.3390/md18020126
[40]	SUN H H, CAO R, LI L H, et al. Cloning, purification and characterization of a novel GH46 family chitosanase, Csn-CAP, from Staphylococcus capitis[J]. Process Biochem, 2018, 75: 146-151. doi: 10.1016/j.procbio.2018.09.021
[41]	鲁晶娣, 韦盘秋, 张兴猛, 等. 壳聚糖酶的研究进展[J]. 中国调味品, 2018, 43(12): 168-173. doi: 10.3969/j.issn.1000-9973.2018.12.033
[42]	LIANG T W, CHEN W T, LIN Z H, et al. An amphiprotic novel chitosanase from Bacillus mycoides and its application in the production of chitooligomers with their antioxidant and anti-inflammatory evaluation[J]. Int J Mol Sci, 2016, 17: 1302. doi: 10.3390/ijms17081302
[43]	马帅, 杨绍青, 刘翊昊, 等. 枯草芽孢杆菌壳聚糖酶在毕赤酵母中的高效表达及其酶解特性[J]. 食品科学, 2019, 40(14): 99-106. doi: 10.7506/spkx1002-6630-20180809-086
[44]	任晶, 赵华, 王虹, 等. 壳聚糖酶的分离纯化及其特性研究[J]. 天津科技大学学报, 2010, 25(6): 10-13. doi: 10.3969/j.issn.1672-6510.2010.06.003
[45]	AZEVEDO M, OLIVEIRA S T, SILVA C, et al. Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides[J]. Int J Biol Macromol, 2020, 165: 1482-1495. doi: 10.1016/j.ijbiomac.2020.09.221

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Identification of key amino acid sites for pH stability of GH46 family chitosanase

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Identification of key amino acid sites for pH stability of GH46 family chitosanase

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