热紫链霉菌几丁质酶表达及低脱乙酰度壳寡糖制备
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摘要
壳寡糖具有抗炎、抗肿瘤及激活植物免疫等一系列生物活性.与传统的高脱乙酰度壳寡糖相比,低脱乙酰度壳寡糖可能具有更高生物活性.表达热紫链霉菌几丁质酶基因并使用表达产物水解制备低脱乙酰度壳寡糖;优化并全基因合成热紫链霉菌几丁质酶基因,利用毕赤酵母进行分泌表达,对产物酶的性质进行鉴定;利用表达的几丁质酶水解制备低脱乙酰度壳寡糖,使用超高效液相色谱-四极杆飞行时间质谱(Ultra-performance liquid chromatography quadrupoletime-of-f lightmassspectrometry,UPLC-QTOFMS)对其组分进行分离及鉴定,使用核磁共振(Nuclear magnetic resonance,NMR)确定其末端结构特征.结果显示,表达的几丁质酶蛋白浓度为0.20 mg/mL,最适pH为5.6,最适温度为60℃,酶活为0.98 U/mL,该酶在80℃及以下时较稳定,该酶水解制备的低脱乙酰度壳寡糖中包含至少35种聚合度2-17、不同脱乙酰度的壳寡糖组分,这些组分的还原末端主要由N-乙酰氨基葡萄糖组成,非还原末端同时包含氨基葡萄糖及N-乙酰氨基葡萄糖.本研究表明毕赤酵母分泌表达的热紫链霉菌几丁质酶具有较好的热稳定性,具有应用于低脱乙酰度壳寡糖规模制备的潜力.(图4表1参22)
Chitooligosaccharides have a range of biological activities, such as anti-inflammatory, anti-tumor, and activation of plant immunity. Compared with traditional high deacetylated chitooligosaccharides, low deacetylated chitooligosaccharides may have higher biological activity. In this study, the chitinase encoding gene of Streptomyces thermoviolaceus was expressed and chitooligosaccharides with a low degree of deacetylation were prepared by the expressed enzyme. The chitinase encoding gene of S. thermoviolaceus was optimized, synthesized, and secretorily expressed in Pichia pastoris. The properties of the produced enzyme were identified. Chitooligosaccharides with a low degree of deacetylation were prepared by the expressed enzyme and the composition and structural characteristics were analyzed by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance, respectively. The protein concentration of the expressed product reached 0.20 mg/mL. The optimum pH and temperature of the expressed chitinase was 5.6 and 60℃, respectively, and enzymatic activity reached 0.98 U/mL. The chitinase was continuously thermostable at 80 ℃. These hydrolysates contained at least 35 different kinds of chitooligosaccharides with a degree of polymerization of 2–17 and different degrees of deacetylation. The reducing end of oligosaccharide fractions were mainly composed of N-Acetylglucosamine,whereas both glucosamine and N-Acetylglucosamine were found in the non-reducing end. This study showed that the chitinase of S. thermoviolaceus secretorily expressed in P. pastoris had good thermal stability and has the potential to be used in the scale preparation of chitooligosaccharides with a low degree of deacetylation.
Chitooligosaccharides have a range of biological activities, such as anti-inflammatory, anti-tumor, and activation of plant immunity. Compared with traditional high deacetylated chitooligosaccharides, low deacetylated chitooligosaccharides may have higher biological activity. In this study, the chitinase encoding gene of Streptomyces thermoviolaceus was expressed and chitooligosaccharides with a low degree of deacetylation were prepared by the expressed enzyme. The chitinase encoding gene of S. thermoviolaceus was optimized, synthesized, and secretorily expressed in Pichia pastoris. The properties of the produced enzyme were identified. Chitooligosaccharides with a low degree of deacetylation were prepared by the expressed enzyme and the composition and structural characteristics were analyzed by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance, respectively. The protein concentration of the expressed product reached 0.20 mg/mL. The optimum pH and temperature of the expressed chitinase was 5.6 and 60℃, respectively, and enzymatic activity reached 0.98 U/mL. The chitinase was continuously thermostable at 80 ℃. These hydrolysates contained at least 35 different kinds of chitooligosaccharides with a degree of polymerization of 2–17 and different degrees of deacetylation. The reducing end of oligosaccharide fractions were mainly composed of N-Acetylglucosamine,whereas both glucosamine and N-Acetylglucosamine were found in the non-reducing end. This study showed that the chitinase of S. thermoviolaceus secretorily expressed in P. pastoris had good thermal stability and has the potential to be used in the scale preparation of chitooligosaccharides with a low degree of deacetylation.
引文
1 Lodhi G,Kim YS,Hwang JW,Kim SK,Jeon YJ,Je JY,Ahn CB,Moon SH,Jeon BT,Park P J.Chitooligosaccharide and its derivatives:preparation and biological applications[J].Biomed Res Int,2014,2014(1):654913
2 Azuma K,Osaki T,Minami S,Okamoto Y.Anticancer and antiinflammatory properties of chitin and chitosan oligosaccharides[J].JFunct Biomater,2015,6(1):33-49
3 Shinya T,Nakagawa T,Kaku H,Shibuya N.Chitin-mediated plantfungal interactions:catching,hiding and handshaking[J].Curr Opin Plant Biol,2015,26:64-71
4 Je JY,Kim SK.Chitooligosaccharides as potential nutraceuticals:production and bioactivities[J].Adv Food Nutr Res,2012,65:321-336
5 Zou P,Yang X,Wang J,Li YF,Yu HL,Zhang YX,Liu GY.Advances in characterization and biological activities of chitosan and chitosan oligosaccharides[J].Food Chem,2016,190:1174-1181
6 Han Y,Zhao L,Yu Z,Feng J,Yu Q.Role of mannose receptor in oligochitosan-mediated stimulation of macrophage function[J].Int Immunopharmacol,2005,5(10):1533-1542
7 Hayafune M,Berisio R,Marchetti R,Silipo A,Kayama M,Desaki Y,Arima S,Squeglia F,Ruggiero A,Tokuyasu K,Molinaro A,Kaku H,Shibuya N.Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization[J].PNAS,2014,111(3):E404-E413
8 Liu B,Li JF,Ao Y,Qu J,Li Z,Su J,Zhang Y,Liu J,Feng D,Qi K,He Y,Wang J,Wang HB.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
9 Xu J,Wang G,Wang J,Li Y,Tian L,Wang X,Guo W.The lysin motifcontaining proteins,Lyp1,Lyk7 and LysMe3,play important roles in chitin perception and defense against Verticillium dahliae in cotton[J].BMC Plant Biol,2017,17(1):148
10 Tegl G,Ohlknecht C,Vielnascher R,Rollett A,Hofinger-Horvath A,Kosma P,Guebitz GM.Cellobiohydrolases produce different oligosaccharides from chitosan[J].Biomacromolecules,2016,17(6):2284-2292
11 Roncal T,Oviedo A,Lopez de Armentia I,Fernandez L,Villaran MC.High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan[J].Carbohydr Res,2007,342(18):2750-2756
12 Lee DX,Xia WS,Zhang JL.Enzymatic preparation of chitooligosaccharides by commercial lipase[J].Food Chem,2008,111(2):291-295
13赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析[J].生物工程学报,2000,16(3):308-311[Zhao X,Huo KK,Li YY.Synonymous codon usage in Pichia pastoris[J].Chin J Biotechnol,2000,16(3):308-311]
14 Chen X,Zhai C,Kang L,Li C,Yan H,Zhou Y,Yu X,Ma L.High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris[J].Biotechnol Lett,2012,34(4):689-694
15 Sasaki C,Varum KM,Itoh Y,Tamoi M,Fukamizo T.Rice chitinases:suga r re cog n it ion sp e ci f icit ie s of t he i nd iv idu al subsit e s[J].Glycobiology,2006,16(12):1242-1250
16 Tsujibo H,Hatano N,Endo H,Miyamoto K,Inamori Y.Purification and characterization of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520 and cloning of the encoding gene[J].Biosci Biotechnol Biochem,2000,64(1):96-102
17 Tsujibo H,Endo H,Minoura K,Miyamoto K,Inamori Y.Cloning and sequence analysis of the gene encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520[J].Gene,1993,134(1):113-117
18 Tsujibo H,Endo H,Miyamoto K,Inamori Y.Expression in Escherichia coli of a gene encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520[J].Biosci Biotechnol Biochem,1995,59(1):145-146
19 Christodoulou E,Duffner F,Vorgias CE.Overexpression,purification,and character izat ion of a ther most able chit i nase(Chi40)f rom Streptomyces thermoviolaceus OPC-520[J].Protein Expr Purif,2001,23(1):97-105
20 Tsujibo H,Okamoto T,Hatano N,Miyamuto K,Watanabe T,Mitsucom M,Inamori Y.Family 19 chitinases from Streptomyces thermoviolaceus OPC-520:molecular cloning and characterization[J].Biosci Biotechnol Biochem,2000,64(11):2445-2453
21 Tews I,Terwisscha van Scheltinga AC,Perrakis A,Wilson KS,Dijkstra BW.Substrate-assisted catalysis unifies two families of chitinolytic enzymes[J].J Am Chem Soc,1997,119(34):7954-7959
22 Sorbot ten A,Hor n SJ,Eijsin k VG,Var um K M.Deg radation of chitosans with chitinase B from Serratia marcescens.production of chito-oligosaccharides and insight into enzyme processivity[J].FEBS J,2005,272(2):538-549
2 Azuma K,Osaki T,Minami S,Okamoto Y.Anticancer and antiinflammatory properties of chitin and chitosan oligosaccharides[J].JFunct Biomater,2015,6(1):33-49
3 Shinya T,Nakagawa T,Kaku H,Shibuya N.Chitin-mediated plantfungal interactions:catching,hiding and handshaking[J].Curr Opin Plant Biol,2015,26:64-71
4 Je JY,Kim SK.Chitooligosaccharides as potential nutraceuticals:production and bioactivities[J].Adv Food Nutr Res,2012,65:321-336
5 Zou P,Yang X,Wang J,Li YF,Yu HL,Zhang YX,Liu GY.Advances in characterization and biological activities of chitosan and chitosan oligosaccharides[J].Food Chem,2016,190:1174-1181
6 Han Y,Zhao L,Yu Z,Feng J,Yu Q.Role of mannose receptor in oligochitosan-mediated stimulation of macrophage function[J].Int Immunopharmacol,2005,5(10):1533-1542
7 Hayafune M,Berisio R,Marchetti R,Silipo A,Kayama M,Desaki Y,Arima S,Squeglia F,Ruggiero A,Tokuyasu K,Molinaro A,Kaku H,Shibuya N.Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization[J].PNAS,2014,111(3):E404-E413
8 Liu B,Li JF,Ao Y,Qu J,Li Z,Su J,Zhang Y,Liu J,Feng D,Qi K,He Y,Wang J,Wang HB.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
9 Xu J,Wang G,Wang J,Li Y,Tian L,Wang X,Guo W.The lysin motifcontaining proteins,Lyp1,Lyk7 and LysMe3,play important roles in chitin perception and defense against Verticillium dahliae in cotton[J].BMC Plant Biol,2017,17(1):148
10 Tegl G,Ohlknecht C,Vielnascher R,Rollett A,Hofinger-Horvath A,Kosma P,Guebitz GM.Cellobiohydrolases produce different oligosaccharides from chitosan[J].Biomacromolecules,2016,17(6):2284-2292
11 Roncal T,Oviedo A,Lopez de Armentia I,Fernandez L,Villaran MC.High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan[J].Carbohydr Res,2007,342(18):2750-2756
12 Lee DX,Xia WS,Zhang JL.Enzymatic preparation of chitooligosaccharides by commercial lipase[J].Food Chem,2008,111(2):291-295
13赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析[J].生物工程学报,2000,16(3):308-311[Zhao X,Huo KK,Li YY.Synonymous codon usage in Pichia pastoris[J].Chin J Biotechnol,2000,16(3):308-311]
14 Chen X,Zhai C,Kang L,Li C,Yan H,Zhou Y,Yu X,Ma L.High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris[J].Biotechnol Lett,2012,34(4):689-694
15 Sasaki C,Varum KM,Itoh Y,Tamoi M,Fukamizo T.Rice chitinases:suga r re cog n it ion sp e ci f icit ie s of t he i nd iv idu al subsit e s[J].Glycobiology,2006,16(12):1242-1250
16 Tsujibo H,Hatano N,Endo H,Miyamoto K,Inamori Y.Purification and characterization of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520 and cloning of the encoding gene[J].Biosci Biotechnol Biochem,2000,64(1):96-102
17 Tsujibo H,Endo H,Minoura K,Miyamoto K,Inamori Y.Cloning and sequence analysis of the gene encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520[J].Gene,1993,134(1):113-117
18 Tsujibo H,Endo H,Miyamoto K,Inamori Y.Expression in Escherichia coli of a gene encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520[J].Biosci Biotechnol Biochem,1995,59(1):145-146
19 Christodoulou E,Duffner F,Vorgias CE.Overexpression,purification,and character izat ion of a ther most able chit i nase(Chi40)f rom Streptomyces thermoviolaceus OPC-520[J].Protein Expr Purif,2001,23(1):97-105
20 Tsujibo H,Okamoto T,Hatano N,Miyamuto K,Watanabe T,Mitsucom M,Inamori Y.Family 19 chitinases from Streptomyces thermoviolaceus OPC-520:molecular cloning and characterization[J].Biosci Biotechnol Biochem,2000,64(11):2445-2453
21 Tews I,Terwisscha van Scheltinga AC,Perrakis A,Wilson KS,Dijkstra BW.Substrate-assisted catalysis unifies two families of chitinolytic enzymes[J].J Am Chem Soc,1997,119(34):7954-7959
22 Sorbot ten A,Hor n SJ,Eijsin k VG,Var um K M.Deg radation of chitosans with chitinase B from Serratia marcescens.production of chito-oligosaccharides and insight into enzyme processivity[J].FEBS J,2005,272(2):538-549