枯草芽孢杆菌壳聚糖酶水解制备低脱乙酰度壳寡糖及其组分分析
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摘要
对来源于枯草芽孢杆菌菌株168(Bacillus subtilis 168)的壳聚糖酶编码基因进行了序列优化及全合成,并在毕赤酵母(Pichia pastoris)中实现了分泌表达,表达产物的蛋白质浓度达到0.30mg/ml。表达的壳聚糖酶最适p H为5.6,最适温度为55℃,比酶活达84.54U/ml。该酶在50℃及以下较稳定。利用该酶水解低脱乙酰度壳聚糖并使用超高效液相色谱-四极杆飞行时间质谱(ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry,UPLC-QTOF MS)对产物的组分进行了分离及鉴定。根据一级质谱信息,推测酶解产物中包含至少37种聚合度2~18,不同脱乙酰度的壳寡糖组分。综上,利用毕赤酵母分泌表达了来源于枯草芽孢杆菌菌株168的壳聚糖酶基因,利用表达产物水解制备了低脱乙酰度壳寡糖并对其组分进行了分析,可为后续壳寡糖结构与功能关系的研究提供参考。
Chitosanase encoding gene of Bacillus subtilis 168 was optimized,synthesized and secretorily expressed in Pichia pastoris. The protein concentration of the expressed product reached 0. 30 mg/ml. The optimum p H and temperature of the expressed chitosanase was 5. 6 and 55℃,respectively,and enzymatic activity reached 84. 54 U/ml. The chitosanase was continuously thermostable at 50℃. The low deacetylated chitosan was hydrolyzed by this enzyme and the composition of these products were analyzed through utraperformance liquid chromatography quadrupole time-of-flight mass spectrometry( UPLC-QTOF MS). The results showed that these hydrolysates contained at least 37 different kinds of chitooligosaccharides with degree of polymerization of 2-18 and different degree of deacetylation. In summary,chitooligosaccharides with low degree of deacetylation were prepared through Bacillus subtilis 168 chitosanase expressed in Pichia pastoris and its composition analyzed,which can provide a reference for the study of the relationship between the structure and function of chitooligosaccharides.
Chitosanase encoding gene of Bacillus subtilis 168 was optimized,synthesized and secretorily expressed in Pichia pastoris. The protein concentration of the expressed product reached 0. 30 mg/ml. The optimum p H and temperature of the expressed chitosanase was 5. 6 and 55℃,respectively,and enzymatic activity reached 84. 54 U/ml. The chitosanase was continuously thermostable at 50℃. The low deacetylated chitosan was hydrolyzed by this enzyme and the composition of these products were analyzed through utraperformance liquid chromatography quadrupole time-of-flight mass spectrometry( UPLC-QTOF MS). The results showed that these hydrolysates contained at least 37 different kinds of chitooligosaccharides with degree of polymerization of 2-18 and different degree of deacetylation. In summary,chitooligosaccharides with low degree of deacetylation were prepared through Bacillus subtilis 168 chitosanase expressed in Pichia pastoris and its composition analyzed,which can provide a reference for the study of the relationship between the structure and function of chitooligosaccharides.
引文
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[25]Xu J,Wang G,Wang J,et al.The lysin motif-containing proteins,Lyp1,Lyk7 and Lys Me3,play important roles in chitin perception and defense against Verticillium dahliae in cotton.BMC Plant Biology,2017,17(1):148.
[26]Liu T,Liu Z,Song C,et al.Chitin-induced dimerization activates a plant immune receptor.Science,2012,336(6085):1160-1164.
[27]Winkler A J,Dominguez-Nunez J A,Aranaz I,et al.Short-chain chitin oligomers:promoters of plant growth.Mar Drugs,2017,15:40.
[2]Zou P,Yang X,Wang J,et al.Advances in characterization and biological activities of chitosan and chitosan oligosaccharides.Food Chemistry,2016,190:1174-1181.
[3]Azuma K,Osaki T,Minami S,et al.Anticancer and antiinflammatory properties of chitin and chitosan oligosaccharides.Journal of Functional Biomaterials,2015,6(1):33-49.
[4]Shinya T,Nakagawa T,Kaku H,et al.Chitin-mediated plantfungal interactions:catching,hiding and handshaking.Current Opinion in Plant Biology,2015,26:64-71.
[5]Je J Y,Kim S K.Chitooligosaccharides as potential nutraceuticals:production and bioactivities.Adv Food Nutr Res,2012,65:321-336.
[6]Li K,Xing R,Liu S,et al.Advances in preparation,analysis and biological activities of single chitooligosaccharides.Carbohydrate Polymers,2016,139:178-190.
[7]Younes I,Rinaudo M.Chitin and chitosan preparation from marine sources.structure,properties and applications.Marine Drugs,2015,13(3):1133-1174.
[8]Jung W J,Park R D.Bioproduction of chitooligosaccharides:present and perspectives.Mar Drugs,2014,12(11):5328-5356.
[9]Tegl G,Ohlknecht C,Vielnascher R,et al.Cellobiohydrolases produce different oligosaccharides from chitosan.Biomacromolecules,2016,17(6):2284-2292.
[10]Ranok A,Wongsantichon J,Robinson R C,et al.High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan.Carbohydr Res,2007,342(18):2750-2756.
[11]Lee D X,Xia W S,Zhang J L.Enzymatic preparation of chitooligosaccharides by commercial lipase.Food Chemistry,2008,111(2):291-295.
[12]中科荣信(苏州)生物科技有限公司.一种里氏木霉几丁质酶及其制备方法和应用:中国,2017106305187.2017-07-28[2018-05-15].http://cpquery.sipo.gov.cn/txn Query Ordinary Patents.Zhongke Runxin(Suzhou)Biological Technology Co.Ltd.Preparation method and application of Trichoderma reesei chitinase:Chinese,2017106305187.2017-07-28[2018-05-15].http://cpquery.sipo.gov.cn/txn Query Ordinary Patents.
[13]Borriss R,Danchin A,Harwood C R,et al.Bacillus subtilis,the model Gram-positive bacterium:20 years of annotation refinement.Microb Biotechnol,2018,11(1):3-17.
[14]程功,任立世,焦思明,等.纤维素酶制备低脱乙酰度壳寡糖及其组成分析.食品科技,2018,43(4):189-193.Cheng G,Ren L S,Jiao S M,et al.Composition analysis and production of chito-oligosanccharides with low degree of deacetylation by cellulase.Food Sience and Technology,2018,43(4):189-193.
[15]Parro V,SanRoman M,Galindo I,et al.A 23 911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the lev operon.Microbiology,1997,143:1321-1326.
[16]Rivas L A,Parro V,Moreno-Paz M,et al.The Bacillus subtilis168 csn gene encodes a chitosanase with similar properties to a streptomyces enzyme.Microbiology,2000,146:2929-2936.
[17]Pechsrichuang P,Yoohat K,Yamabhai M.Production of recombinant Bacillus subtilis chitosanase,suitable for biosynthesis of chitosan-oligosaccharides.Bioresour Technol,2013,127:407-414.
[18]Kang L X,Chen X M,Fu L,et al.Recombinant expression of chitosanase from Bacillus subtilis HD145 in Pichia pastoris.Carbohydr Res,2012,352:37-43.
[19]Feng J,Zhao L,Yu Q.Receptor-mediated stimulatory effect of oligochitosan in macrophages.Biochemical and Biophysical Research Communications,2004,317(2):414-420.
[20]Han Y,Zhao L,Yu Z,et al.Role of mannose receptor in oligochitosan-mediated stimulation of macrophage function.International Immunopharmacology,2005,5(10):1533-1542.
[21]Schimpl M,Rush C L,Betou M,et al.Human YKL-39 is a pseudo-chitinase with retained chitooligosaccharide-binding properties.Biochemical Journal,2012,446(1):149-157.
[22]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).Journal of Biological Chemistry,2015,290(5):2617-2629.
[23]Hayafune M,Berisio R,Marchetti R,et al.Chitin-induced activation of immune signaling by the rice receptor CEBi P relies on a unique sandwich-type dimerization.Proceedings of the National Academy of Sciences,2014,111(3):E404-E413.
[24]Liu B,Li J F,Ao Y,et al.Lysin motif-containing proteins LYP4and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity.Plant Cell,2012,24(8):3406-3419.
[25]Xu J,Wang G,Wang J,et al.The lysin motif-containing proteins,Lyp1,Lyk7 and Lys Me3,play important roles in chitin perception and defense against Verticillium dahliae in cotton.BMC Plant Biology,2017,17(1):148.
[26]Liu T,Liu Z,Song C,et al.Chitin-induced dimerization activates a plant immune receptor.Science,2012,336(6085):1160-1164.
[27]Winkler A J,Dominguez-Nunez J A,Aranaz I,et al.Short-chain chitin oligomers:promoters of plant growth.Mar Drugs,2017,15:40.