食品与发酵工业 >

2019 , Vol. 45 >Issue 3: 77 - 82

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.018288

研究报告

海洋来源褐藻胶裂解酶及其基因B1SM的克隆和表达

  • 吴晨烁 ,
  • 李晓月 ,
  • 秦明珍 ,
  • 严芬
展开
  • (福州大学 生物科学与工程学院,福建 福州,350108)
硕士研究生(严芬副教授为通讯作者,E-mail:yanfen@fzu.edu.cn)。

收稿日期: 2018-07-16

  网络出版日期: 2019-03-11

基金资助

福建省财政厅项目(闽财指(2015)1297号);福建省省财政厅项目(闽财指(2014)1262号);福州市市级科技计划项目(2017X0035);校科技发展基金(2014-XQ-20);校科技发展基金(2013-XQ-28)

收起

Cloning and expression of the alginate lyase gene B1SM frommarine Pseudoalteromonas sp.

  • B1WU Chenshuo ,
  • LI Xiaoyue ,
  • QIN Mingzhen ,
  • YAN Fen
Expand
  • (College of Biological Science and Technology, Fuzhou University, Fuzhou 350108,China)

Received date: 2018-07-16

  Online published: 2019-03-11

Fold

摘要

从海洋来源的假交替单胞菌(Pseudoalteromonas sp.)B1基因组中克隆1个褐藻胶裂解酶基因,并在大肠杆菌中实现异源表达,分离纯化重组酶并研究其酶学性质。通过Touch down PCR与热不对称交错PCR(thermal asymmetric interlaced PCR, TAIL-PCR)从假交替单胞菌B1基因组中扩增到1个褐藻胶裂解酶基因(B1SM),将目的基因插入到pGEX-4T-1载体,转化到宿主大肠杆菌(Escherichia coli,E. coli)BL21 (DE3)。结果显示,从菌株B1克隆得到褐藻胶裂解酶基因B1SM,序列长度为1 005 bp,编码334个氨基酸,理论等电点为8.51,编码蛋白质的理论分子质量为37.13 kDa。重组褐藻胶裂解酶B1SM的最适pH和温度分别为8.0和25 ℃。该褐藻胶裂解酶在pH 7.0~9.0范围内,25 ℃下保温1 h,仍剩余60%以上活力,pH稳定性较好。在最适pH 8.0和30 ℃下保温1 h重组酶仍剩余60%以上活力。该研究为褐藻胶裂解酶基因B1SM在E. coli BL21(DE3)中实现了异源表达,为褐藻胶裂解酶的制备和研究奠定基础。

关键词： 褐藻胶裂解酶; 假交替单胞菌; 聚合酶链反应

本文引用格式

吴晨烁 , 李晓月 , 秦明珍 , 严芬 . 海洋来源褐藻胶裂解酶及其基因B1SM的克隆和表达[J]. 食品与发酵工业, 2019 , 45(3) : 77 -82 . DOI: 10.13995/j.cnki.11-1802/ts.018288

Abstract

The recombinant alginate lyase B1SM was isolated and purified, and its enzymatic properties were characterized. The alginate lyase gene B1SM was amplified from the genome of Pseudomonas aeruginosa B1 by Touch down polymerase chain reaction (PCR) and Thermal Asymmetric Interlaced PCR (TAIL-PCR). The gene B1SM was inserted into a pGEX-4T-1 expression vector and expressed in Escherichia coli BL21 (DE3). The results showed that the optimal pH and temperature for B1SM were 8.0 and 25℃, respectively. Moreover, B1SM maintained more than 60% of its relative enzyme activity after incubating at 25℃ for 1 h at pH=7.0-9.0, indicating that it had a good pH stability. Furthermore, it maintained its relative enzyme activity to more than 60% after incubating at 30 ℃ and at pH=8.0 for 1 h. It was concluded that heterologous expression of alginate lyase gene B1SM was achievable in E. coli BL21 (DE3), which laid a foundation for preparing and further study of alginate lyase.

Key words： alginate lyase; Pseudoalteromonas sp.; polymerase chain reaction (PCR)

参考文献

[1] 罗丹丹,薛永常. 褐藻胶裂解酶的研究进展[J]. 生物学杂志, 2016, 33(6): 95-98.
[2] VERA J,CASTRO J, GONZALEZ A,et al. Seaweed polysaccharides and derived oligosaccharides stimulate defense responses and protection against pathogens in plants[J]. Marine Drugs, 2011, 9(12): 2 514.
[3] WANG D,YUN E J,KIM S,et al. Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae[J]. Bioprocess Biosyst Eng, 2016, 39(6): 959-966.
[4] MARÍA C R,RICARDO P C,SEBASTIAN V S,et al. Comparison of different types of pretreatment and enzymatic saccharification of Macrocystis pyrifera for the production of biofuel[J]. Algal Research, 2016, 13: 141-147.
[5] 王雪松,郑芸,方积年. 降血糖多糖及寡糖的研究进展[J]. 药学学报, 2004, 39(12): 1 028-1 033.
[6] 王庭欣,赵文,蒋东升,等. 海带多糖对小鼠免疫功能的调节作用[J]. 毒理学杂志, 2000, 14(2):75.
[7] LIU H,GENG M,XIN X,et al. Multiple and multivalent interactions of novel anti-AIDS drug candidates, sulfated polymannuronate (SPMG)-derived oligosaccharides, with gp120 and their anti-HIV activities[J]. Glycobiology, 2005, 15(5): 501-510.
[8] GENG Meiyu,LI Fuchuan,XIN Xianliang,et al. The potential molecular targets of marine sulfated polymannuroguluronate interfering with HIV-1 entry: Interaction between SPMG and HIV-1 rgp120 and CD4 molecule[J]. Antiviral Research, 2003, 59(2): 127-135.
[9] MAMI IWAMOTO,MAKI KURACHI,TAKUJI NAKASHIMA,et al. Structure–activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells[J]. Febs Letters, 2005, 579(20): 4 423-4 429.
[10] YOSHIKO I, XU X U,TADASHI T,et al. Enzymatically depolymerized alginate oligomers that cause cytotoxic cytokine production in human mononuclear cells[J]. Bioscience, Biotechnology and Biochemistry, 2003,67(2):258-263.
[11] MACDONALD L C,BERGER B W. A polysaccharide lyase from Stenotrophomonas maltophilia with a unique, pH-regulated substrate specificity[J]. Journal of Biological Chemistry, 2014, 289(1): 312-325.
[12] ZHU B,YIN H. Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications[J]. Bioengineered, 2015, 6(3): 125-131.
[13] TAKAGI T,YOKOI T,SHIBATA T,et al. Engineered yeast whole-cell biocatalyst for direct degradation of alginate from macroalgae and production of non-commercialized useful monosaccharide from alginate[J]. Applied Microbiology and Biotechnology, 2016, 100(4): 1 723-1 732.
[14] 魏丹,窦文芳,李恒,等.高效降解褐藻胶新菌种的筛选、鉴定及产酶条件优化[J]. 食品与发酵工业, 2012, 38(7): 26-31.
[15] 李恒,朱思婷,刘旭梅,等. 褐藻胶裂解酶产生菌的分离鉴定及产酶发酵优化[J]. 中国生物工程杂志, 2014, 34(9): 94-101.
[16] MOHAPATRA B R. Kinetic and thermodynamic properties of alginate lyase and cellulase co-produced by Exiguobacterium species Alg-S5[J]. Internationl Journal of Biological Macromolecules, 2017, 98: 103-110.
[17] ZHU Y,WU L,CHEN Y,et al. Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates[J]. Microbiological Research, 2016, 182: 49-58.
[18] 严芬,连燕萍,杨光,等. 高产褐藻胶裂解酶菌株的筛选及发酵条件优化[J]. 食品工业科技, 2015, 36(22).
[19] HIRAYAMA M,HASHIMOTO W,MURATA K,et al. Comparative characterization of three bacterial exo-type alginate lyases[J]. Internationl Journal of Biological Macromolecules, 2016, 86: 519-524.
[20] MATHIEU S,HENRISSAT B,LABRE F,et al. Functional exploration of the polysaccharide lyase family PL6[J]. PLoS One, 2016, 11(7): e0 159 415.
[21] ZHU B,TAN H,QIN Y,et al. Characterization of a new endo-type alginate lyase from Vibrio sp. W13[J]. Internationl Journal of Biological Macromolecules, 2015, 75: 330-337.
[22] LIU Y,SAKAI S,TAYA M. Engineering tissues with a perfusable vessel-like network using endothelialized alginate hydrogel fiber and spheroid-enclosing microcapsules[J]. Heliyon, 2016, 2(2): e00067.
[23] LI S,WANG L,HAN F,et al. Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp. Kz7[J]. Journal of Biological Chemistry, 2016, 159(1): 77-86.
[24] LI S,YANG X,ZHANG L,et al. Cloning, expression, and characterization of a cold-adapted and surfactant-stable alginate lyase from marine bacterium Agarivorans sp. L11[J]. Journal of Microbiology and Biotechnology, 2015, 25(5): 681-686.
[25] YANG X,LI S,WU Y,et al. Cloning and characterization of two thermo- and salt-tolerant oligoalginate lyases from marine bacterium Halomonas sp.[J]. FEMS Microbiology Letters, 2016, 363(9).
[26] CHEN X L,DONG S,XU F,et al. Characterization of a new cold-adapted and salt-activated polysaccharide lyase family 7 alginate lyase from Pseudoalteromonas sp. SM0524[J]. Front Microbiol, 2016, 7: 1 120.
[27] 刘航,曹海龙,岳敏,等. 褐藻胶裂解酶基因的克隆、表达载体构建以及表达条件的研究[J]. 华中师范大学学报(自科版), 2012, 46(4): 456-460.
[28] PARK D,JAGTAP S,NAIR S K. Structure of a PL17 family alginate lyase demonstrates functional similarities among exotype depolymerases[J]. Journal of Biological Chemistry, 2014, 289(12): 8 645-8 655.
[29] 严芬,王新,叶秀云. 一株碱性普鲁兰酶菌株的筛选、鉴定及酶学性质研究[J]. 福州大学学报:自然科学版, 2014, 42(4): 633-638.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司