壳聚糖酶的基因克隆表达及酶学性质研究
|
摘要
作者从NCBI数据库中挖掘到来源于Butyrivibrio sp.MC2013的壳聚糖酶基因,命名为BUT,该壳聚糖酶基因大小为903 bp,编码301个氨基酸。通过NCBI数据库和进化树比对发现,该壳聚糖酶(BUT)属糖苷水解酶46家族(后简称GH-46),与其它壳聚糖酶相似度为59%,是一种新型的壳聚糖酶。序列经密码子优化后进行全基因序列合成,与表达载体pET21a(+)连接构建重组质粒p ET-21a-BUT,转入大肠杆菌E.coli BL21(DE3)表达宿主,进行诱导表达。所得重组壳聚糖酶通过Ni-NTA亲和层析进行纯化,SDS-PAGE确定其蛋白分子量为35 kDa,DNS法测定其酶活为146.0 U/mg。对BUT酶的酶学性质进行探究,结果表明BUT酶最适温度和pH分别为45℃、8.0,在中性偏碱性条件下稳定性较强。Mn~(2+)对其酶活力具有促进作用,SDS、Fe~(3+)、Cu~(2+)、Zn~(2+)等的抑制作用极强。通过TLC对其水解产物分析发现,BUT水解壳聚糖的产物是壳二糖、壳三糖和壳四糖。
A chitosanase gene BUT from the Butyrivibrio sp.MC2013 was selected from the NCBI database,which contains 903 bp and encodes a protein with 301 animo acids.Research has suggested that the chitosanase belongs to the GH-46 by sequence alignment in NCBI datebase and phylogenetic analysis.It is a novel chitosanase which shares 59%identities with any other chitosanases.The genes weresynthesized after codon optimization and inserted into pET21a(+),then the recombinant plasmid,pET-21a-BUT,was transformed into E.coli BL21(DE3).The chitosanase was induced in ZYP-5052 and purified with the affinity chromatography of Ni-NTA.The molecular weight was estimated to be 35 kDa by SDS-PAGE.The maximum activity of purified enzyme was 146.0 U/mg.The enzymatic properties of recombinant chitosanase showed that the optimal temperature and pH of the BUT were 45℃ and 8.0,respectively.The stability in alkaline conditions was higher than that in acidic conditions.Mn~(2+)could significantly enhance the enzymatic activity,while SDS,Fe~(3+),Cu~(2+)and Zn~(2+)could inhibit the activity strongly.The analysis of hydrolysates by TLC demonstrated that chitosan was degraded into(GlcN)_2,(GlcN)_3,and(GlcN)_4.
A chitosanase gene BUT from the Butyrivibrio sp.MC2013 was selected from the NCBI database,which contains 903 bp and encodes a protein with 301 animo acids.Research has suggested that the chitosanase belongs to the GH-46 by sequence alignment in NCBI datebase and phylogenetic analysis.It is a novel chitosanase which shares 59%identities with any other chitosanases.The genes weresynthesized after codon optimization and inserted into pET21a(+),then the recombinant plasmid,pET-21a-BUT,was transformed into E.coli BL21(DE3).The chitosanase was induced in ZYP-5052 and purified with the affinity chromatography of Ni-NTA.The molecular weight was estimated to be 35 kDa by SDS-PAGE.The maximum activity of purified enzyme was 146.0 U/mg.The enzymatic properties of recombinant chitosanase showed that the optimal temperature and pH of the BUT were 45℃ and 8.0,respectively.The stability in alkaline conditions was higher than that in acidic conditions.Mn~(2+)could significantly enhance the enzymatic activity,while SDS,Fe~(3+),Cu~(2+)and Zn~(2+)could inhibit the activity strongly.The analysis of hydrolysates by TLC demonstrated that chitosan was degraded into(GlcN)_2,(GlcN)_3,and(GlcN)_4.
引文
[1]YANG Jiulin,XIE Hongguo,YU Weiting,et al.Research progress in chitosan for tissue engineering[J].Functional Materials,2013,44(11):1521-1525.(in Chinese)
[2]THADATHIL N,VELAPPAN S P.Recent developments in chitosanase research and its biotechnological applications:A review[J].Food Chemistry,2014,150(1):392-399.
[3]KIM S K,RAJAPAKSE N.Enzymatic production and biological activities of chitosan oligosaccharides(COS):A review[J].Carbohydrate Polymers,2005,62(4):357-368.
[4]BALAN V,VERESTIUC L.Strategies to improve chitosan hemocompatibility:A review[J].European Polymer Journal,2014,53(1):171-188.
[5]CHEN X,XIA W,YU X.Purification and characterization of two types of chitosanase from Aspergillus,sp.CJ22-326[J].Food Research International,2005,38(3):315-322.
[6]LI Songlin.Cloning,expression and site-directed mutagenesis of chitosanase gene produced by Aspergillus CJ22-326[D].Jiangnan University,2009.
[7]LU Huading,LIAN Liyi,CHEN Mingwei,et al.Cloning of Aspergillus gene and its expression in Escherichia Coli[J].Chinese Journal of Tissue Engineering Research,2014,18(34):5490-5496.(in Chinese)
[8]ZHENG Fuchun.Cloning,expression and enzymatic analysis of chitosanase gene from Bacillus subtilis[D].East China University of Technology,2017.(in Chinese)
[9]ZHANG J,CAO H,LI S,et al.Characterization of a new family 75 chitosanase from Aspergillus sp.W-2[J].International Journal of Biological Macromolecules,2015,81(NOV):362-369.
[10]NIDHEESH T,PAL G K,SURESH P V.Chitooligomers preparation by chitosanase produced under solid state fermentation using shrimp by-products as substrate[J].Carbohydrate Polymers,2015,121:1-9.
[11]PECHSRICHUANG P,YOOHAT K,YAMABHAI M.Production of recombinant Bacillus subtilis chitosanase,suitable for biosynthesis of chitosan-oligosaccharides[J].Bioresource Technology,2013,127(127C):407-414.
[12]LIU Wanshun,WANG Jiao,YANG Yan,et al.Expression of chitosanase gene in Yarrowia lipolytica and recombinase properties[J].Journal of Ocean University of China,2011,41(12):58-62.(in Chinese)
[13]KURITA K.Chemistry and application of chitin and chitosan[J].Polymer Degradation&Stability,1998,59(1-3):117-120.
[14]PECHSRICHUANG P,LORENTZEN S B,AAM B B,et al.Bioconversion of chitosan into chito-oligosaccharides(CHOS)using family 46 chitosanase from Bacillus subtilis(Bs Csn46A)[J].Carbohydrate Polymers,2018,186:420-428.
[15]PASCAL V,MARIE-EVE L H,RYSZARD B.Chitosanases from family 46 of glycoside hydrolases:From proteins to phenotypes[J].Marine Drugs,2015,13(11):6566-6587.
[16]YANG Jülin.Preparation and analysis of chitosanase separation,purification,properties and degradation products chito-oligosaccharides[D].Ocean University of China,2005.(in Chinese)
[17]KIM P I,KANG T H,CHUNG K J,et al.Purification of a constitutive chitosanase produced by Bacillus sp.MET 1299 with cloning and expression of the gene[J].Fems Microbiology Letters,2010,240(1):31-39.
[18]ZOU P,YANG X,WANG J,et al.Advances in characterisation and biological activities of chitosan and chitosan oligosaccharides[J].Food Chemistry,2016,190:1174-1181.
[19]SUN Beibei.Development and application of key enzymes for the recycling of crustacean waste[D].Harbin Institute of Technology,2016.(in Chinese)
[20]XIA W,LIU P,LIU J.Advance in chitosan hydrolysis by non-specific cellulases[J].Bioresource Technology,2008,99(15):6751-6762.
[21]LQianqian.Study on substrate binding and catalytic mechanism of chitosanase OU01[D].Ocean University of China,2015.(in Chinese)
[2]THADATHIL N,VELAPPAN S P.Recent developments in chitosanase research and its biotechnological applications:A review[J].Food Chemistry,2014,150(1):392-399.
[3]KIM S K,RAJAPAKSE N.Enzymatic production and biological activities of chitosan oligosaccharides(COS):A review[J].Carbohydrate Polymers,2005,62(4):357-368.
[4]BALAN V,VERESTIUC L.Strategies to improve chitosan hemocompatibility:A review[J].European Polymer Journal,2014,53(1):171-188.
[5]CHEN X,XIA W,YU X.Purification and characterization of two types of chitosanase from Aspergillus,sp.CJ22-326[J].Food Research International,2005,38(3):315-322.
[6]LI Songlin.Cloning,expression and site-directed mutagenesis of chitosanase gene produced by Aspergillus CJ22-326[D].Jiangnan University,2009.
[7]LU Huading,LIAN Liyi,CHEN Mingwei,et al.Cloning of Aspergillus gene and its expression in Escherichia Coli[J].Chinese Journal of Tissue Engineering Research,2014,18(34):5490-5496.(in Chinese)
[8]ZHENG Fuchun.Cloning,expression and enzymatic analysis of chitosanase gene from Bacillus subtilis[D].East China University of Technology,2017.(in Chinese)
[9]ZHANG J,CAO H,LI S,et al.Characterization of a new family 75 chitosanase from Aspergillus sp.W-2[J].International Journal of Biological Macromolecules,2015,81(NOV):362-369.
[10]NIDHEESH T,PAL G K,SURESH P V.Chitooligomers preparation by chitosanase produced under solid state fermentation using shrimp by-products as substrate[J].Carbohydrate Polymers,2015,121:1-9.
[11]PECHSRICHUANG P,YOOHAT K,YAMABHAI M.Production of recombinant Bacillus subtilis chitosanase,suitable for biosynthesis of chitosan-oligosaccharides[J].Bioresource Technology,2013,127(127C):407-414.
[12]LIU Wanshun,WANG Jiao,YANG Yan,et al.Expression of chitosanase gene in Yarrowia lipolytica and recombinase properties[J].Journal of Ocean University of China,2011,41(12):58-62.(in Chinese)
[13]KURITA K.Chemistry and application of chitin and chitosan[J].Polymer Degradation&Stability,1998,59(1-3):117-120.
[14]PECHSRICHUANG P,LORENTZEN S B,AAM B B,et al.Bioconversion of chitosan into chito-oligosaccharides(CHOS)using family 46 chitosanase from Bacillus subtilis(Bs Csn46A)[J].Carbohydrate Polymers,2018,186:420-428.
[15]PASCAL V,MARIE-EVE L H,RYSZARD B.Chitosanases from family 46 of glycoside hydrolases:From proteins to phenotypes[J].Marine Drugs,2015,13(11):6566-6587.
[16]YANG Jülin.Preparation and analysis of chitosanase separation,purification,properties and degradation products chito-oligosaccharides[D].Ocean University of China,2005.(in Chinese)
[17]KIM P I,KANG T H,CHUNG K J,et al.Purification of a constitutive chitosanase produced by Bacillus sp.MET 1299 with cloning and expression of the gene[J].Fems Microbiology Letters,2010,240(1):31-39.
[18]ZOU P,YANG X,WANG J,et al.Advances in characterisation and biological activities of chitosan and chitosan oligosaccharides[J].Food Chemistry,2016,190:1174-1181.
[19]SUN Beibei.Development and application of key enzymes for the recycling of crustacean waste[D].Harbin Institute of Technology,2016.(in Chinese)
[20]XIA W,LIU P,LIU J.Advance in chitosan hydrolysis by non-specific cellulases[J].Bioresource Technology,2008,99(15):6751-6762.
[21]LQianqian.Study on substrate binding and catalytic mechanism of chitosanase OU01[D].Ocean University of China,2015.(in Chinese)