解淀粉芽孢杆菌草酸脱羧酶基因的克隆、原核表达与活力测定
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摘要
为进一步明确解淀粉芽孢杆菌C(10)的草酸脱羧酶基因的生物功能,克隆其关键基因对该基因进行原核表达。通过PCR技术克隆了淀粉芽孢杆菌C(10)的草酸脱羧酶基因,构建了原核生物表达载体pET28a-Baoxdc,对其进行表达与纯化。结果表明,扩增所获解淀粉芽孢杆菌C(10)的草酸脱羧酶全基因序列全长为1 161 bp,在大肠杆菌中进行表达,添加诱导剂IPTG、MnCl_2至终浓度分别为0.6、6 mmol/L,诱导4 h时的酶活力和比活力最高,分别为3.793 2 U/mL和3.145 3 U/mg。
引文
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[2]杨胜远,韦锦,李云,等.一株产抗菌活性物质解淀粉芽孢杆菌的筛选及鉴定[J].食品科学,2010,31(21):208-212.
[3]Bajaj I,Singhal R.Poly(glutamic acid)-an emerging biopolymer of commercial interest[J].Bioresource Technology,2011,102(10):5551-5561.
[4]Ashiuchi M,Shimanouchi K,Nakamura H,et al.Enzymatic synthesis of high molecular masspoly gamma glutamate and regulation of its stereochemistry[J].Applied and Environmental Microbiology,2004,70(7):4249-4255.
[5]Bovarnick M.The formation of extracellular D(—)glutamic acid polypeptide by Bacillus subtilis[J].Biological Chemistry,1942(145):415-424.
[6]Just V J,Stevenson C M,Bowater L,et al.A closed conformation of Bacillus subtilis oxalate decarboxylase Oxd C provides evidence for the true identity of the active site[J].Journal of Biological Chemistry,2004,279(19):19867-19874.
[7]贺俊斌,林日辉,韦成昱,等.草酸脱羧酶的性质及应用研究进展[J].食品科学,2015,36(1):262-267.
[8]赵树田,张士青.草酸代谢酶的研究进展[J].上海交通大学学报(医学版),2007,27(10):1274-1277.
[9]Cassland P,Sjode A,Winestrand S,et al.Evaluation of oxalate decarboxylase and oxalate oxidase for industrial applications[J].Applied Biochemistry and Biotechnology,2010,161(1/2/3/4/5/6/7/8):255-263.
[10]Makela M R,Hilden K,Lundell T K.Oxalate decarboxylase:biotechnological update and prevalence of the enzyme in filamentous fungi[J].Applied Microbiology and Biotechnology,2010,87(3):801-814.
[11]于俊杰,陈志谊,胡建坤,等.枯草芽孢杆菌Bs-916草酸脱羧酶基因Bacisubin的克隆、原核表达及其表达产物的酶活性分析[J].江苏农业学报,2012,28(3):497-502.
[12]Twahir U T,Stedwell C N,Lee C T,et al.Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4[J].Free Radical Biology and Medicine,2015,80:59-66.
[13]Sasikumar P,Gomathi S,Anbazhagan K,et al.Secretion of biologically active heterologous oxalate decarboxylase (OxdC) in Lactobacillus plantarum WCFS1 using homologous signal peptides[J/OL].BioMed Research International,2013,2013:9(2013-07-18).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732618/.
[14]Svedruzic D,Liu Y,Reinhardt L A,et al.Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis[J].Archives of Biochemistry and Biophysics,2007,464(1):36-47.