强烈炽热球菌几丁质酶水解制备低脱乙酰度壳寡糖及其组成与结构分析
详细信息    查看全文 | 推荐本文 |
  • 英文篇名:Preparation of Chitooligosaccharides with Low Degree of Deacetylation by Recombinant Chitinase from Pyrococcus furiosus, Its Compositions and Structure Analysis
  • 作者:程功 ; 焦思明 ; 任立世 ; 冯翠 ; 康跻耀 ; 杜昱光
  • 英文作者:CHENG Gong;JIAO Siming;REN Lishi;FENG Cui;KANG Jiyao;DU Yuguang;State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences;
  • 关键词:强烈炽热球菌 ; 几丁质酶 ; 壳寡糖 ; 分子排阻高效液相色谱 ; 基质辅助激光解吸电离飞行时间质谱 ; 核磁共振
  • 英文关键词:Pyrococcus furiosus;;chitinase;;chitooligosaccharides;;size exclusion high performance liquid chromatography (SE-HPLC);;matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS);;nuclear magnetic resonance(NMR)
  • 中文刊名:SPKX
  • 英文刊名:Food Science
  • 机构:中国科学院过程工程研究所生化工程国家重点实验室;
  • 出版日期:2017-12-12 15:40
  • 出版单位:食品科学
  • 年:2018
  • 期:v.39;No.583
  • 基金:“十三五”国家重点研发计划重点专项(2016YFB0301502);; 国家自然科学基金联合基金项目(U1608255)
  • 语种:中文;
  • 页:SPKX201818018
  • 页数:5
  • CN:18
  • ISSN:11-2206/TS
  • 分类号:122-126
摘要
利用全基因合成方法合成了强烈炽热球菌(Pyrococcus furiosus)的几丁质酶编码基因并在大肠杆菌(Escherichia coli)中实现了可溶表达。利用该酶对低脱乙酰度壳聚糖进行水解并对获得的壳寡糖产物进行组成及结构分析。分子排阻高效液相色谱结果显示,水解产物相对分子质量分布范围为1 000~5 000。基质辅助激光解吸电离飞行时间质谱分析结果显示,酶解产物中包含聚合度2~9、不同脱乙酰度的壳寡糖。核磁共振对酶解产物壳寡糖的结构鉴定结果显示,所有寡糖组分的还原端均主要由两个连续的N-乙酰氨基葡萄糖组成。综上,本研究利用来源于强烈炽热球菌的几丁质酶制备了还原末端结构确定的低脱乙酰度壳寡糖,为复杂结构壳寡糖结构与功能关系研究提供了理论支持。
        The chitinase-encoding gene of Pyrococcus furiosus was synthesized and the recombinant enzyme solubly expressed in Escherichia coli. Chitosan with low degree of deacetylation was hydrolyzed by this enzyme and the composition and structure of the resulting chitooligosaccharides were analyzed. Size exclusion high performance liquid chromatography(SE-HPLC) results showed that the relative molecular masses of the hydrolysis products ranged from 1 000 to 5 000. Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS) analysis revealed that the hydrolysates contained chitooligosaccharides with a degree of polymerization of 2-9 and different degrees of deacetylation. Structural characterization by nuclear magnetic resonance(NMR) indicated that the reducing end of all oligosaccharide fractions was mainly composed of two successive N-acetylglucosamines. In summary, we have prepared chitooligosaccharides with low degree deacetylation having determined reducing end structure using recombinant chitinasefrom P. furiosus, which can lay the foundation for the study of the structural-activity relationship of chitooligosaccharides with complex structures.
引文
[1]LODHI G,KIM Y S,HWANG J W,et al.Chitooligosaccharide and its derivatives:preparation and biological applications[J].Biomed Research International,2014,2014:65491 3.DOI:10.1155/2014/654913.
    [2]LI K,XING R,LIU S,et al.Advances in preparation,analysis and biological activities of single chitooligosaccharides[J].Carbohydrate Polymers,2016,139:178-190.DOI:10.1016/j.carbpol.2015.12.016.
    [3]ZOU P,YANG X,WANG J,et al.Advances in characterization and biological activities of chitosan and chitosan oligosaccharides[J].Food Chemistry,2016,190:1174-1181.DOI:10.1016/j.foodchem.2015.06.076.
    [4]WINKLER A J,DOMINGUEZ-NUNEZ J A,ARANAZ I,et al.Shortchain chitin oligomers:promoters of plant growth[J].Marine Drugs,2017,15(2):40.DOI:10.3390/md15020040.
    [5]JE J Y,KIM S K.Chitooligosaccharides as potential nutraceuticals:production and bioactivities[J].Advances in Food and Nutrition Research,2012,65:321-336.DOI:10.1016/b978-0-12-416003-3.00021-4.
    [6]KERCH G.The potential of chitosan and its derivatives in prevention and treatment of age-related diseases[J].Marine Drugs,2015,13(4):2158-2182.DOI:10.3390/md13042158.
    [7]FENG J,ZHAO L,YU Q.Receptor-mediated stimulatory effect of oligochitosan in macrophages[J].Biochemical and Biophysical Research Communications,2004,317(2):414-420.DOI:10.1016/j.bbrc.2004.03.048.
    [8]HAN Y,ZHAO L,YU Z,et al.Role of mannose receptor in oligochitosan-mediated stimulation of macrophage function[J].International Immunopharmacology,2005,5(10):1533-1542.DOI:10.1016/j.intimp.2005.04.015.
    [9]SCHIMPL M,RUSH C L,BETOU M,et al.Human YKL-39 is a pseudo-chitinase with retained chitooligosaccharide-binding properties[J].Biochemical Journal,2012,446(1):149-157.DOI:10.1042/bj20120377.
    [10]EINARSSON J M,BAHRKE S,SIGURDSSON B T,et al.Partially acetylated chitooligosaccharides bind to YKL-40 and stimulate growth of human osteoarthritic chondrocytes[J].Biochemical and Biophysical Research Communications,2013,434(2):298-304.DOI:10.1016/j.bbrc.2013.02.122.
    [11]RANOK A,WONGSANTICHON J,ROBINSON R C,et al.Structural and thermodynamic insights into chitooligosaccharide binding to human cartilage chitinase 3-like protein 2(CHI3L2 or YKL-39)[J].Journal of Biological Chemistry,2015,290(5):2617-2629.DOI:10.1074/jbc.M114.588905.
    [12]CAROTENUTO G,CHABAUD M,MIYATA K,et al.The rice LysMreceptor-like kinase OsCERK1 is required for the perception of shortchain chitin oligomers in arbuscular mycorrhizal signaling[J].New Phytologist,2017,214(4):1440-1446.DOI:10.1111/nph.14539.
    [13]HAYAFUNE M,BERISIO R,MARCHETTI R,et al.Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization[J].Proceedings of the National Academy of Sciences,2014,111(3):E404-E413.DOI:10.1073/pnas.1312099111.
    [14]LIU B,LI J F,AO Y,et al.Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity[J].Plant Cell,2012,24(8):3406-3419.DOI:10.1105/tpc.112.102475.
    [15]XU J,WANG G,WANG J,et al.The lysin motif-containing proteins,Lyp1,Lyk7 and LysMe3,play important roles in chitin perception and defense against Verticillium dahliae in cotton[J].BMC Plant Biology,2017,17(1):148.DOI:10.1186/s12870-017-1096-1.
    [16]GUST A A,WILLMANN R,DESAKI Y,et al.Plant LysM proteins:modules mediating symbiosis and immunity[J].Trends in Plant Science,2012,17(8):495-502.DOI:10.1016/j.tplants.2012.04.003.
    [17]KELLY S,RADUTOIU S,STOUGAARD J.Legume LysM receptors mediate symbiotic and pathogenic signalling[J].Current Opinion in Plant Biology,2017,39:152-158.DOI:10.1016/j.pbi.2017.06.013.
    [18]YOUNES I,RINAUDO M.Chitin and chitosan preparation from marine sources.Structure,properties and applications[J].Marine Drugs,2015,13(3):1133-1174.DOI:10.3390/md13031133.
    [19]JUNG W J,PARK R D.Bioproduction of chitooligosaccharides:present and perspectives[J].Marine Drugs,2014,12(11):5328-5356.DOI:10.3390/md12115328.
    [20]XIE Y,HU J,WEI Y,et al.Preparation of chitooligosaccharides by the enzymatic hydrolysis of chitosan[J].Polymer Degradation&Stability,2009,94(10):1895-1899.DOI:10.1016/j.polymdegradstab.2009.06.021.
    [21]LEE D X,XIA W S,ZHANG J L.Enzymatic preparation of chitooligosaccharides by commercial lipase[J].Food Chemistry,2008,111(2):291-295.DOI:10.1016/j.foodchem.2008.03.054.
    [22]RONCAL T,OVIEDO A,LóPEZ DE ARMENTIA I,et al.High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan[J].Carbohydrate Research,2007,342(18):2750-2756.DOI:10.1016/j.carres.2007.08.023.
    [23]陈季旺,谭晶,夏文水.木瓜蛋白酶中具有水解壳聚糖活性的酶组分分离纯化[J].食品科学,2009,30(17):235-238.
    [24]平向莉,江波,张涛等.纤维素酶制备壳寡糖工艺研究[J].食品工业科技,2011,32(7):263-266.
    [25]OKU T,ISHIKAWA K.Analysis of the hyperthermophilic chitinase from Pyrococcus furiosus:activity toward crystalline chitin[J].Bioscience,Biotechnology,and Biochemistry,2006,70(7):1696-1701.DOI:10.1271/bbb.60031.
    [26]KURITA K,SANNAN T,IWAKURA Y.Studies on chitin,.4.Evidence for formation of block and random copolymers of N-acetyl-D-glucosamine and D-glucosamine by heterogeneous and homogeneous hydrolyses[J].Makromolekulare ChemieMacromolecular Chemistry and Physics,1977,178(12):3197-3202.
    [27]LAVERTU M,XIA Z,SERREQI A N,et al.A validated 1H NMRmethod for the determination of the degree of deacetylation of chitosan[J].Journal of Pharmaceutical and Biomedical Analysis,2003,32(6):1149-1158.DOI:10.1016/S0731-7085(03)00155-9.
    [28]SASAKI C,VARUM K M,ITOH Y,et al.Rice chitinases:sugar recognition specificities of the individual subsites[J].Glycobiology,2006,16(12):1242-1250.DOI:10.1093/glycob/cwl043.
    [29]NAKAMURA T,MINE S,HAGIHARA Y,et al.Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus[J].Acta Crystallographica Section F-Structural Biology and Crystallization Communications,2007,63:7-11.DOI:10.1107/s1744309106051773.
    [30]SORBOTTEN A,HORN S J,EIJSINK V G,et al.Degradation of chitosans with chitinase B from Serratia marcescens.Production of chito-oligosaccharides and insight into enzyme processivity[J].FEBS Journal,2005,272(2):538-549.DOI:10.1111/j.1742-4658.2004.04495.x.

© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号

地址:北京市海淀区学院路29号 邮编:100083

电话:办公室:(+86 10)66554848;文献借阅、咨询服务、科技查新:66554700