Abstract
Objective: This study focused on improving the production of chitosan oligosaccharides by chitinase degradation of chitin. Methods: Using genetic engineering methods to clone, prokaryotic expressing, and investigating enzymatic properties of the chitinase from Streptomyces diastaticus, and the chitinase activity was explored by homology modeling, amino acid comparison of the catalytic domain, and site-directed mutagenesis. Results: After cloning and prokaryotic expression, the enzyme production cycle was shortened from 7 d to 24 h, and the enzyme activity reached 132 U/L, which was 32% higher than the original bacterial enzyme activity (100 U/L). The amino acids that determine the activity of chitinase was Asp at position 128, 130, and Glu at position 132 of the catalytic domain. Conclusion: Clonal expression of the chitinase gene resulted in a shorter enzyme production cycle and increased enzyme activity.
Publication Date
6-30-2022
First Page
1
Last Page
7,70
DOI
10.13652/j.spjx.1003.5788.2022.90090
Recommended Citation
Qin, LI; Li-mei, WANG; and Bin, QI
(2022)
"Cloning, expression and catalytic function analysis of chitinase from Streptomyces diastaticus,"
Food and Machinery: Vol. 38:
Iss.
5, Article 1.
DOI: 10.13652/j.spjx.1003.5788.2022.90090
Available at:
https://www.ifoodmm.cn/journal/vol38/iss5/1
References
[1] THARANATHAN R N,KITTUR F S.Chitin-The undisputed biomolecule of great potential[J].Critical Reviews in Food Science and Nutrition,2003,43(1):61-87.
[2] HORN S J,SIKORSKI P,CEDERKVIST J B,et al.Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides[J].P Natl Acad Sci USA,2006,103(48):18 089-18 094.
[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.WU Yu-xiao,XU Hai-tao,GAO Yun-hua,et al.Research progress on production of chitooligosaccharides[J].The Science and Technology of Gelatin,2015,35(3):128-132.
[4] THAMTHIANKUL S,SUAN-NGAY S,TANTIMAVANICH S,et al.Chitinase from Bacillus thuringiensis subsp Pakistani[J].Appl Microbiol Biotechnol,2001,27(56):395-401.
[5] WEI Guo-guang,ZHANG A-lei,CHEN Ke-quan,et al.Enzymatic production of N-acetyl-D-glucosamine from crayfish shell wastes pretreated via high pressure homogenization[J].Carbohydrate Polymers,2017,37(171):236-241.
[6] TAIRA T,FUJIWARA M,DENNHART N,et al.Transglycosylation reaction catalyzed by a class V chitinase from cycad,Cycas revoluta:A study involving site-directed mutagenesis,HPLC,and real-time ESI-MS[J].BBA Proteins Proteom,2010,1 804(4):668-675.
[7] WANG S L,LIN T Y,YEN Y H,et al.Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase[J].Carbohydrate Research,2006,341(15):2 507-2 515.
[8] WANG Di,LI An-jie,HAN Hong-yu,et al.A potent chitinase from Bacillus subtilis for the efficient bioconversion of chitin-containing wastes[J].International Journal of Biological Macromolecules,2018,40(116):863-868.
[9] MALLAKUNTLA M K,VAIKUNTAPU P R,BHUVANACHANDR A,et al.Transglycosylation by a chitinase from Enterobacter cloacae subsp.cloacae generates longer chitin oligosaccharides[J].Scientific Reports,2017,7(1):5 113-5 525.
[10] 潘梦妍.甲壳素酶的克隆表达与分子改造研究[D].无锡:江南大学,2019:2.PAN Meng-yan.Cloning,expression and molecular modification of chitinase[D].Wuxi:Jiangnan University,2019:2.
[11] ZAKARIASSEN H,HANSEN M C,JRANLI M,et al.Mutational effects on transglycosylating activity of family 18 chitinases and construction of a hypertransglycosylating mutant[J].Biochemistry,2011,50(25):5 693-5 703.
[12] MADHUPRAKASH J,SINGH A,KUMAR S,et al.Singh,structure of chitinase D from Serratia proteamaculans reveals the structural basis of its dual action of hydrolysis and transglycosylation[J].International Journal of Biochemistry & Molecular Biology,2013,4(4):166-178.
[13] LONHIENNE T,MAVROMATIS K,VORGIAS C E,et al.Cloning,sequences,and characterization of two chitinase genes from the Antarctic Arthrobacter sp.strain TAD20:Isolation and partial characterization of the enzymes[J].Journal of Bacteriology,2001,183(5):1 773-1 779.
[14] DONG Hui-zhong,WANG Yao-song,ZHAO Li-ming,et al.Key Technologies of enzymatic preparation for DP 6~8 chitooligosaccharides[J].J Food Eng,2015,38(4):336-344.
[15] 李静,赵祥颖,刘建军.微生物几丁质酶及其在食品领域的应用[J].食品工程,2006,34(1):47-48.LI Jing,ZHAO Xiang-ying,LIU Jian-jun.The review of microbial chitinase and its application in food field[J].Food Engineering,2006,34(1):47-48.
[16] 陈三凤,李季伦.黄杆菌(Flavobacterium sp.)几丁质酶的纯化和性质[J].微生物学报,2005,26(2):14-19.CHEN San-feng,LI Ji-lun.Purification and properties of chitinase from Flavobacterium sp[J].Acta Microbiologica Sinica,2005,26(2):14-19.
[17] 张博阳,朱天辉,韩珊,等.桑氏链霉菌几丁质酶ChiKJ40基因的克隆表达及其抑菌作用[J].微生物学通报,2018,45(5):1 016-1 026.ZHANG Bo-yang,ZHU Tian-hui,HAN Shan,et al.Cloning,expression and antibacterial functions of Chi KJ40,a chitinase gene from Streptomyces sampsonii[J].Microbiology China,2018,45(5):1 016-1 026.
[18] LI Shu-jiang,ZHANG Bo-yang,ZHU Han-ming-yue,et al.Cloning and expression of the chitinase encoded by ChiKJ406136 from streptomyces sampsonii(Millard & Burr)waksman KJ40 and its antifungal effect[J].Forests,2018,9(11):699-717.
[19] LAEMMLI U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227(5 259):680-685.
[20] 连明珠.南极Pseudoalteromonas sp.AC444低温几丁质酶性质分析与基因克隆及普里兹湾深海沉积物中几丁质酶基因多样性研究[D].厦门:厦门大学,2006:28.LIAN Ming-zhu.Characterization and gene clone of cold-active chitinase from the Pseudoalteromonas sp.AC444 and chitinase diversity in deep sea sediment of Prydz Bay,Antarctic[D].Xiamen:Xiamen University,2006:28.
[21] 张学英,黄忠意,章发盛.两种检测还原糖方法的比较[J].食品与机械,2017,33(2):66-69.ZHANG Xue-ying,HUANG Zhong-yi,ZHANG Fa-sheng.Comparative study on two methods for sugar detection in Jerusalem artichoke[J].Food & Machinery,2017,33(2):66-69.
[22] SCHWEDE T,KOPP J,GUEX N,et al.SWISS-MODEL:An automated protein homology-modeling server[J].Nucleic Acids Research,2003,31(13):3 381-3 385.
[23] 郝俊尧,马富强,杨广宇.产碱杆菌Alcaligenes sp.KS-85来源肌酸酶活性中心的关键氨基酸功能研究[J].生物技术通报,2020,37(3):1-9.HAO Jun-yao,MA Fu-qiang,YANG Guang-yu.Functional analysis of key residues in the active center of creatinase from Alcaligenes sp.KS-85[J].Biotechnology Bulletin,2020,37(3):1-9.
[24] CAI Wen-xuan,LI Sha,ZHOU Jia-wei,et al.Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis[J].World Journal of Microbiology & Biotechnology,2009,25(12):2 147-2 155.
[25] SYNSTAD B,GASEIDNES S,VAN AALTEN D M F,et al.Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase[J].Eur J Biochem,2004,271(2):253-262.
[26] DUHMEKLAIR A.Crystal structure of a bacterial chitinase at 2.3 Å resolution[J].Structure,1994,2(12):1 169-1 180.
[27] VAAJE-KOLSTAD G,HOUSTON D R,RAO F V,et al.Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin[J].Bba-Proteins Proteom,2004,1 696(1):103-111.
[28] NI Hong,ZENG Si-quan,QIN Xiao-wei,et al.Molecular docking and site-directed mutagenesis of a bacillus thuringiensis chitinase to improve chitinolytic,synergistic lepidopteran-larvicidal and nematicidal activities[J].International Journal of Biological Sciences,2015,11(3):304-315.
[29] SUGINTA W,SRITHO N.Multiple roles of Asp313 in the refined catalytic cycle of chitin degradation by vibrio harveyi chitinase A[J].Bioscience Biotechnology and Biochemistry,2012,76(12):2 275-2 281.
[30] 王晓辉,曹洪玉,张庆芳,等.低温几丁质酶chiA基因克隆、同源建模及序列分析[J].大连大学学报,2015,36(6):60-65.WANG Xiao-hui,CAO Hong-yu,ZHANG Qing-fang,et al.Cloning,homology modeling and sequecnce analysis of cold-adapted chiA[J].Journal of Dalian University,2015,36(6):60-65.
[31] RAO V P,KRISHNA M M,NARAYAN D S,et al.Applicability of endochitinase of Flavobacterium johnsoniae with transglycosylation activity in generating long-chain chitooligosaccharides[J].International Journal of Biological Macromolecules,2018,40(117):62-71.