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黄桂媛,温顺华,李锋,卢明倩,王巧贞,廖威,黄庶识.Shewanella haliotis BP-1海藻酸裂解酶基因的克隆表达[J].广西科学,2016,23(3):228-233. [点击复制]
- HUANG Guiyuan,WEN Shunhua,LI Feng,LU Mingqian,WANG Qiaozhen,LIAO Wei,HUANG Shushi.Gene Cloning and Expression of Alginate Lyase from Shewanella haliotis BP-1[J].Guangxi Sciences,2016,23(3):228-233. [点击复制]
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| Shewanella haliotis BP-1海藻酸裂解酶基因的克隆表达 |
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黄桂媛1, 温顺华2, 李锋3, 卢明倩1, 王巧贞1, 廖威4, 黄庶识1
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| (1.广西科学院生物物理实验室, 广西南宁 530007;2.厦门万泰凯瑞生物技术有限公司, 福建厦门 361003;3.黔南州民族师范学院化学化工学院, 贵州都匀 558000;4.广西职业技术学院, 广西南宁 530226) |
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| 摘要: |
| [目的]了解海洋细菌Shewanella haliotis BP-1中海藻酸裂解酶降解海藻酸钠的生物活性。[方法]应用基因克隆和大肠杆菌异源表达技术,过量表达海藻酸裂解酶,将粗酶液通过DEAE Sepharose FF柱分离纯化后检测其酶活性。[结果]从S.haliotis BP-1菌株的基因组DNA中克隆得到一个大小为2 157 bp的海藻酸裂解酶基因Alg17S,该基因编码的海藻酸裂解酶Alg17S属于PL17家族的蛋白,大小为79 726 Da,其中包括N端26个氨基酸的信号肽,与Saccharophagus degradans 2-40菌株产生的海藻酸裂解酶Alg17C具有高度同源性,相似性为52%。经纯化后获得的重组酶Alg17S和△snAlg17S (N端不含26个氨基酸的信号肽)均具有降解海藻酸钠的活性,但△snAlg17S对海藻酸钠的催化活性比Alg17S高,其酶比活力高达9 635 U/mg。[结论]重组海藻酸裂解酶△snAlg17S兼具高表达水平及高酶活性,是进一步研究海藻酸盐糖化和生物燃料生产的潜在的优势酶。 |
| 关键词: 海藻酸裂解酶 克隆表达 酶活性测定 Shewanella haliotis BP-1 |
| DOI：10.13656/j.cnki.gxkx.20160713.003 |
| 投稿时间：2016-04-28修订日期：2016-06-17 |
| 基金项目:国家自然科学基金项目(31560017),广西自然科学重点基金项目(2014GXNSFDA118012)和贵州省教育厅自然科学研究重点项目(黔教合KY[2014]286)资助。 |
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| Gene Cloning and Expression of Alginate Lyase from Shewanella haliotis BP-1 |
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HUANG Guiyuan1, WEN Shunhua2, LI Feng3, LU Mingqian1, WANG Qiaozhen1, LIAO Wei4, HUANG Shushi1
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| (1.Lab of Biophysics, Guangxi Academy of Sciences, Nanning, Guangxi, 530007, China;2.Xiamen Innodx Biotech Co. Ltd., Xiamen, Fujian, 361003, China;3.School of Chemistry and Chemical Engineering, Qiannan Normal College for Nationalities, Duyun, Guizhou, 558000, China;4.Guangxi Vocational and Technical College, Nanning, Guangxi, 530226, China) |
| Abstract: |
| [Objective] Alginate lyase in Shewanella haliotis BP-1 strains was studied illustrate its biological activity of degrading alginate.[Methods] The gene cloning technology and the Escherichia coli heterologous expression technology were applied to overexpress the alginate lyase;And the enzyme activity was analyzed after the crude enzyme was separated and purified by DEAE Sepharose FF chromatography.[Results] The alginate lyase gene Alg17S,with a size of 2 157 bp,was cloned from S.haliotis BP-1 strain genomic DNA and encoded an alginate lyase Alg17S,which belonged to polysaccharide lyase(PL)17 family and had a size of 79 726 Da protein(including an N-terminal signal peptide of 26 amino acid signal peptide).Alg17S showed high sequence identity of 52% with PL-17 protein sequence Alg17C from Saccharophagus degradans 2-40.Both the purified recombinase Alg17S and the △snAlg17S(without the N-terminal signal peptide of 26 amino acids) can degrade alginate,but the enzymatic activity of △snAlg17S revealed a specific activity of 9 635 U/mg,which was more efficient than Alg17S.[Conclusion] The recombinant alginate lyase △snAlg17S that has both high-level expression and high enzymatic activity could be a potential enzyme for further researching on the alginate saccharification and the biofuels production. |
| Key words: alginate lyase cloning and expression enzyme activity determination Shewanella haliotis BP-1 |
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