毕赤酵母表达解淀粉芽孢杆菌壳聚糖酶及其水解制备可控结构壳寡糖
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摘要
优化并全合成解淀粉芽孢杆菌(Bacillus amyloliquefaciens)壳聚糖酶编码基因并在毕赤酵母(Pichia pastoris)中实现分泌表达,表达产物的蛋白质量浓度达到0.23 mg/mL。壳聚糖水解酶的最适pH值为5.0,最适温度为45℃,比活力达52.2 U/mL。该酶在50℃以下较稳定。利用该酶水解低脱乙酰度壳聚糖并对产物进行了组成及结构分析。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3～15、不同脱乙酰度的壳寡糖。核磁共振鉴定结果显示,壳寡糖组分的还原末端及非还原末端均主要由氨基葡萄糖组成。综上,本研究高效表达了来源于解淀粉芽孢杆菌的壳聚糖酶,并制备了确定末端结构的壳寡糖,为壳寡糖的结构与功能关系研究提供理论支持。
The chitosanase-encoding gene of Bacillus amyloliquefaciens was optimized, synthesized and expressed in Pichia pastoris. The protein concentration of the expressed product was 0.23 mg/mL. The optimum pH and temperature of the enzyme were 5.0 and 45 ℃, respectively, and the speci?c activity reached 52.2 U/mL. The chitosanase was continuously thermo-stable at 50 ℃. Chitosan with low deacetylation was hydrolyzed by this enzyme and the composition and structure of the hydrolysate were analyzed. Matrix-assisted laser desorption/ionization time of ?ight mass spectrometry(MALDITOF MS) analysis showed that this hydrolysate contained chitooligosaccharides with degree of polymerization of 3–15 and different degrees of deacetylation. Nuclear magnetic resonance(NMR) results indicated that both the reducing and nonreducing ends of oligosaccharide fractions were mainly composed of glucosamine. In summary, we ef?ciently expressed chitosanase from B. amyloliquefaciens and prepared chitooligosaccharides with determined terminal structures, which will lay a theoretical foundation for the study of the structure-function relationship of chitooligosaccharides.
The chitosanase-encoding gene of Bacillus amyloliquefaciens was optimized, synthesized and expressed in Pichia pastoris. The protein concentration of the expressed product was 0.23 mg/mL. The optimum pH and temperature of the enzyme were 5.0 and 45 ℃, respectively, and the speci?c activity reached 52.2 U/mL. The chitosanase was continuously thermo-stable at 50 ℃. Chitosan with low deacetylation was hydrolyzed by this enzyme and the composition and structure of the hydrolysate were analyzed. Matrix-assisted laser desorption/ionization time of ?ight mass spectrometry(MALDITOF MS) analysis showed that this hydrolysate contained chitooligosaccharides with degree of polymerization of 3–15 and different degrees of deacetylation. Nuclear magnetic resonance(NMR) results indicated that both the reducing and nonreducing ends of oligosaccharide fractions were mainly composed of glucosamine. In summary, we ef?ciently expressed chitosanase from B. amyloliquefaciens and prepared chitooligosaccharides with determined terminal structures, which will lay a theoretical foundation for the study of the structure-function relationship of chitooligosaccharides.
引文
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[30]程功,焦思明,任立世,等.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[J].食品科学,2019,39(18):115-119.DOI:10.7506/spkx1002-6630-201818018.
[2]THADATHIL N,VELAPPAN S P.Recent developments in chitosanase research and its biotechnological applications:a review[J].Food Chemistry,2014,150:392-399.DOI:10.1016/j.foodchem.2013.10.083.
[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]AZUMA K,OSAKI T,MINAMI S,et al.Anticancer and antiinflammatory properties of chitin and chitosan oligosaccharides[J].Journal of Functional Biomaterials,2015,6(1):33-49.DOI:10.3390/jfb6010033.
[5]SHINYA T,NAKAGAWA T,KAKU H,et al.Chitin-mediated plant-fungal interactions:catching,hiding and handshaking[J].Current Opinion in Plant Biology,2015,26:64-71.DOI:10.1016/j.pbi.2015.05.032.
[6]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.
[7]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.
[8]TEGL G,OHLKNECHT C,VIELNASCHER R,et al.Cellobiohydrolases produce different oligosaccharides from chitosan[J].Biomacromolecules,2016,17(6):2284-2292.DOI:10.1021/acs.biomac.6b00547.
[9]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.
[10]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.
[11]黄永春,李琳,郭祀远,等.木瓜蛋白酶降解壳聚糖动力学研究[J].食品科学,2003,24(4):34-38.
[12]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.
[13]段静,刘广鑫,董晏君,等.解淀粉芽孢杆菌HZ-1510壳聚糖酶基因的克隆、重组表达及其活性[J].水产学报,2017,41(10):1552-1561.
[14]中科荣信(苏州)生物科技有限公司.一种枯草芽孢杆菌壳聚糖酶及其制备方法和应用:2017106295081[P].2017-07-28.
[15]CHEN X H,KOUMOUTSI A,SCHOLZ R,et al.Comparative analysis of the complete genome sequence of the plant growthpromoting bacterium Bacillus amyloliquefaciens FZB42[J].Nature Biotechnology,2007,25(9):1007-1014.DOI:10.1038/nbt1325.
[16]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.
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[27]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.
[28]LIU T,LIU Z,SONG C,et al.Chitin-induced dimerization activates a plant immune receptor[J].Science,2012,336:1160-1164.DOI:10.1126/science.1218867.
[29]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.
[30]程功,焦思明,任立世,等.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[J].食品科学,2019,39(18):115-119.DOI:10.7506/spkx1002-6630-201818018.