枯草芽孢杆菌壳聚糖酶在毕赤酵母中的高效表达及其酶解特性
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摘要
研究枯草芽孢杆菌壳聚糖酶基因(Bs Csn46)在巴斯德毕赤酵母(Pichia pastoris)GS115中的高效表达、重组酶性质及其酶解特性。重组菌在5 L发酵罐高密度发酵后胞外酶活力高达50 370 U/mL,蛋白质量浓度15.7 mg/mL。粗酶经强阴离子交换层析纯化,纯酶比活力为4 065.7 U/mg,最适pH 6.0,最适温度55℃,在45℃以下保持稳定。该酶水解3 g/100 mL壳聚糖得到主产物为二糖、三糖和四糖的壳寡糖,水解率为92.8%,壳寡糖得率为90.9%。本研究的重组壳聚糖酶产酶水平和水解效率高,为工业化制备壳聚糖酶及大规模制备壳寡糖的应用提供了理论支持。
In this study, the gene encoding chitosanase(BsCsn46) was cloned from Bacillus subtilis and expressed at high level in Pichia pastoris GS115. The enzymatic and hydrolytic properties of BsCsn46 were further investigated.The extracellular chitosanase activity and protein concentration was up to 50 370 U/mL and 15.7 mg/mL after the high cell-density fermentation of the recombinant P. pastoris in a 5 L fermentor, respectively. BsCsn46 was purified by ion-exchange chromatography using Q-Sepharose Fast Flow. The purified enzyme had a high specific activity of 4 065.7 U/mg towards chitosan and showed maximal activity at pH 6.0 and 55 ℃, and it was stable at temperature below 45 ℃. The hydrolysis of 3 g/100 mL chitosan by BsCsn46 yielded predominantly chitooligosaccharides with degree of polymerization(DP) 2–4. The degree of hydrolysis reached 92.8% and the yield of chitooligosaccharides was up to 90.9%.The high enzyme production level and the remarkable hydrolysis efficiency make BsCsn 46 a promising candidate for application in the industrial preparation of chitosanase and chitooligosaccharides.
In this study, the gene encoding chitosanase(BsCsn46) was cloned from Bacillus subtilis and expressed at high level in Pichia pastoris GS115. The enzymatic and hydrolytic properties of BsCsn46 were further investigated.The extracellular chitosanase activity and protein concentration was up to 50 370 U/mL and 15.7 mg/mL after the high cell-density fermentation of the recombinant P. pastoris in a 5 L fermentor, respectively. BsCsn46 was purified by ion-exchange chromatography using Q-Sepharose Fast Flow. The purified enzyme had a high specific activity of 4 065.7 U/mg towards chitosan and showed maximal activity at pH 6.0 and 55 ℃, and it was stable at temperature below 45 ℃. The hydrolysis of 3 g/100 mL chitosan by BsCsn46 yielded predominantly chitooligosaccharides with degree of polymerization(DP) 2–4. The degree of hydrolysis reached 92.8% and the yield of chitooligosaccharides was up to 90.9%.The high enzyme production level and the remarkable hydrolysis efficiency make BsCsn 46 a promising candidate for application in the industrial preparation of chitosanase and chitooligosaccharides.
引文
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[27]YANG Z L,ZHANG Z S.Engineering strategies for enhanced production of protein and bio-products in Pichia pastor is:a review[J].Biotechnology Advances,2017,36(1):182-195.DOI:10.1016/j.biotechadv.2017.11.002.
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[29]POTVIN G,AHMAD A,ZHANG Z S.Bioprocess engineering aspects of heterologous protein production in Pichia pastoris:a review[J].Biochemical Engineering Journal,2012,64(19):91-105.DOI:10.1016/j.bej.2010.07.017.
[30]杨光,盛军,陈亚东,等.枯草芽孢杆菌壳聚糖酶基因在毕赤酵母中的表达及酶学性质[J].中国水产科学,2017,24(6):1288-1297.DOI:10.3724/SP J.1 1 18.201 7.17 129.
[31]CHOIJ Y J,KIM E J,PIAO Z,et al.Purification and characterization of chitosanase from Bacillus sp.strain KCTC 0377BP and its application for the production of chitosan oligosaccharides[J].Applied&Environmental Microbiology,2004,70(8):4522-4531.DOI:10.1 128/AEM.70.8.4522-453 1.2004.
[32]SEVO M,DEGRASSI G,SKOKO N,et al.Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris[J].FEMS Yeast Research,2002,1:271-277.DOI:10.1111/j.1567-1364.2002.tb00045.x.
[33]GAO X G,ZHANG Y F,PARK R D,et al.Preparation of chitooligosaccharides from chitosan using crude enzyme of Bacillus cereus D-11[J].Journal of Applied Biological Chemistry,2012,55(1):13-17.DOI:10.3839/j abc.201 1.053.
[34]CHENG C H,LI Y K.An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides[J].Biotechnology and Applied Biochemistry,2000,32(3):197-203.
[2]LIAQAT F,ELTEM R.Chitooligosaccharides and their biological activities:a comprehensive review[J].Carbohydrate Polymers,2018,184:243-259.DOI:10.1016/j.carbpol.2017.12.067.
[3]THADATHIL N,VELAPPAN S P.Recent developments in chitosanase research and its biotechnological applications:a review[J].Food Chemistry,2014,150(1):392-399.DOI:10.1016/j.foodchem.2013.10.083.
[4]龚香艺,吴静,邬敏辰.真菌壳聚糖酶研究进展[J].食品科学,2012,33(17):308-311.
[5]虞凤慧,徐泽平,谢海涛.一株产壳聚糖酶芽孢杆菌的分离及鉴定[J].中国酿造,20 15,34(1):65-67.DOI:10.1 1882/j.issn.0254-507 1.2015.01.016.
[6]冯志彬,薛钰,陈国忠,等.1株产壳聚糖酶细菌的分离、鉴定和发酵条件优化[J].食品科学,2016,37(19):171-176.DOI:10.7506/spkx1002-6630-20 1619029.
[7]DENIS F,BRZEZINSKI R.An improved aminoglycoside resistance gene cassette for use in Gram-negative bacteria and Streptomyces[J].FEMS Microbiology Letters,1991,81(3):261-264.
[8]LIN J S,CKEN C C,HU H Y.Homologous and heterologous expression of chitosanase gene from Bacillus subtilis NCHU-05[J].Journal of Biotechnology,2010,150:S354-S354.DOI:1 0.1016/j.jbiotec.2010.09.400.
[9]PECHSRICHUANG P,YOOHAT K,YAMABHAI M.Production of recombinant Bacillus subtilis chitosanase,suitable for biosynthesis of chitosan-oligosaccharides[J].Bioresource Technology,2013,127:407-414.DOI:10.1016/j.biortech.2012.09.130.
[10]PENG N,XU W L,WANG F,et al.Mitsuaria chitosanase with unrevealed important amino acid residues:characterization and enhanced production in Pichia pastoris[J].Applied Microbiology and Biotechnology,2013,97(1):171-179.DOI:10.1016/j.biortech.2012.09.130.
[11]ZHOU Z P,ZHAO S Z,WANG S Q,et al.Extracellular overexpression of chitosanase from Bacillus sp.TS in Escherichia coli[J].Applied Biochemistry and Biotechnology,2015,175(7):3271-3286.DOI:10.1007/s00253-0 12-3901-y.
[12]SU P C,HSUEH W C,CHANG W S,et al.Enhancement of chitosanase secretion by Bacillus subtilis for production of chitosan oligosaccharides[J].Journal of the Taiwan Institute of Chemical Engineers,2017,29:49-54.DOI:10.1016/j.j tice.2016.12.040.
[13]KANG L X,JIANG S J,MA L X,Enzymatic production of high molecular weight chitooligosaccharides using recombinant chitosanase from Bacillus thuringiensis BMB 171[J].Microbiology Biotechnology Letters,2018,46(1):45-50.DOI:10.4014/mbl.1712.12012.
[14]QIN Z,CHEN Q M,LIN S,et al.Expression and characterization of a novel cold-adapted chitosanase suitable for chitooligosaccharides controllable preparation[J].Food Chemistry,2018,253:139-147.DOI:10.101 6/j.foodchem.201 8.01.137.
[15]SUN H H,MAO X Z,GUO N,et al.Discovery and characterization of a novel chitosanase from Paenibacillus dendritiformis by phylogenybased enzymatic product specificity prediction[J].Journal of Agricultural Food Chemistry,2018,66(1 8):4645-465 1.DOI:10.1021/acs.jafc.7b06067.
[16]CHEN X M,ZHAI C,KANG L X,et al.High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris[J].Biotechnology Letters,2012,34(4):689-694.DOI:10.1007/s 10529-01 1-08 16-0.
[17]HUANG L,WANG Q H,JIANG S J,et al.Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus fumigatus in Escherichia coli[J].Bioprocess and Biosystems Engineering,2016,39(1 1):1-9.DOI:10.1007/s00449-016-1643-4.
[18]RIVAS L A,PARRO V,MORENO-PAZ M,et al.The Bacillus subtilis 168 csn gene encodes a chitosanase with similar properties to a Streptomyces enzyme[J].Microbiology,2000,146(1 1):2929-2936.
[19]KANG L X,CHEN X M,FU L,et al.Recombinant expression of chitosanase from Bacillus subtilis HD145 in Pichia pastoris[J].Carbohydrate Research,2012,352(5):37-43.DOI:10.1016/j.carres.2012.01.025.
[20]MOURYA V K,INAMDAR N N,CHOUDHARI Y M.Chitooligosaccharides:synthesis,characterization and applications[J].Polymer Science,20 1 1,53(7):583-612.DOI:10.1134/S0965545X11070066.
[21]CABRERA J C,CUTSEM P V.Preparation of chitooligosaccharides with degree of polymerization higher than 6 by acid or enzymatic degradation of chitosan[J].Biochemical Engineering Journal,2005,25(2):165-172.DOI:10.101 6/j.bej.2005.04.025.
[22]JIANG Z Q,WEI Y,LI D Y,et al.High-level production,purification and characterization of a thermostableβ-mannanase from the newly isolated Bacillus subtilis WY-34[J].Carbohydrate Polymers,2006,66(1):88-96.DOI:10.101 6/j.carbpol.2006.02.030.
[23]张舒平,周鹏,苏春元,等.重组枯草芽孢杆菌壳聚糖酶的纯化和性质研究[J].中国农业大学学报,2012,17(1):125-131.
[24]MILLER G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical Biochemistry,1959,3 1(3):426-428.
[25]LOWRY O H,ROSEBROUGH N J,FARR A L,et al.Protein measurement with the folin phenol reagent[J].Journal of Biological Chemistry,1951,193(1):265-275.
[26]LAEMMLI U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227:680-685.
[27]YANG Z L,ZHANG Z S.Engineering strategies for enhanced production of protein and bio-products in Pichia pastor is:a review[J].Biotechnology Advances,2017,36(1):182-195.DOI:10.1016/j.biotechadv.2017.11.002.
[28]JIA H Y,FAN G S,YAN Q J,et al.High-level expression of a hyperthermostable Thermotoga maritime xylanase in Pichia pastoris by codon optimization[J].Journal of Molecular Catalysis B Enzymatic,2012,78:72-77.DOI:10.1016/j.molcatb.2012.02.009.
[29]POTVIN G,AHMAD A,ZHANG Z S.Bioprocess engineering aspects of heterologous protein production in Pichia pastoris:a review[J].Biochemical Engineering Journal,2012,64(19):91-105.DOI:10.1016/j.bej.2010.07.017.
[30]杨光,盛军,陈亚东,等.枯草芽孢杆菌壳聚糖酶基因在毕赤酵母中的表达及酶学性质[J].中国水产科学,2017,24(6):1288-1297.DOI:10.3724/SP J.1 1 18.201 7.17 129.
[31]CHOIJ Y J,KIM E J,PIAO Z,et al.Purification and characterization of chitosanase from Bacillus sp.strain KCTC 0377BP and its application for the production of chitosan oligosaccharides[J].Applied&Environmental Microbiology,2004,70(8):4522-4531.DOI:10.1 128/AEM.70.8.4522-453 1.2004.
[32]SEVO M,DEGRASSI G,SKOKO N,et al.Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris[J].FEMS Yeast Research,2002,1:271-277.DOI:10.1111/j.1567-1364.2002.tb00045.x.
[33]GAO X G,ZHANG Y F,PARK R D,et al.Preparation of chitooligosaccharides from chitosan using crude enzyme of Bacillus cereus D-11[J].Journal of Applied Biological Chemistry,2012,55(1):13-17.DOI:10.3839/j abc.201 1.053.
[34]CHENG C H,LI Y K.An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides[J].Biotechnology and Applied Biochemistry,2000,32(3):197-203.