来源于假单孢菌206植酸酶基因的克隆、表达及酶学性质研究

生物技术通报 ›› 2013, Vol. 0 ›› Issue (8): 139-144.

来源于假单孢菌206植酸酶基因的克隆、表达及酶学性质研究

李雅楠¹, 黄火清², 姚斌², 赵青¹, 刘昆¹, 马文康¹

（1.广州医学院生物化学与分子生物学教研室，广州 510182；2.中国农业科学院饲料研究所，北京 100081）

收稿日期:2013-08-11 修回日期:2013-08-11 出版日期:2013-08-11 发布日期:2013-09-02
作者简介:李雅楠，女，博士，讲师，研究方向：微生物工程；E-mail：yanan_2007@126.com
基金资助:
广州医学院科研项目（2006GD058）

Gene Cloning，Expression and Characterization of an HAP Phytase from Pseudomonas fluorescens 206

Li Yanan¹, Huang Huoqing², Yao Bin², Zhao Qing¹, Liu Kun¹, Ma Wenkang¹

（1. Depatment of Biochemistry，Guangzhou Medical College，Guangzhou 510182；2. Feed Research Institute，Chinese Academy of Agricultural Sciences, Beijing 100081）

Received:2013-08-11 Revised:2013-08-11 Published:2013-08-11 Online:2013-09-02

摘要/Abstract

摘要： 从浙江嘉兴人工鱼塘底泥样品筛选到产植酸酶活性较高的假单孢菌(Pseudomonas fluorescens)206。通过简并PCR和TAIL-PCR技术从Pseudomonas fluorescens 206菌株基因组DNA中克隆得到一个新的编码组氨酸酸性植酸酶（HAP）的基因，命名为phyP。该基因ORF全长1 284 bp，编码了427个氨基酸和一个终止密码子，前24个氨基酸为信号肽。将phyP在大肠杆菌中表达，重组蛋白经硫酸铵分级沉淀和阴离子交换柱纯化后达到电泳纯。对其酶学性质分析表明：重组植酸酶PhyP最适pH为5.5，在pH3.5-7.5的条件下具有较好的稳定性；重组植酸酶PhyP最适温度为45℃，在25℃时也具有较高的酶活性，因此植酸酶PhyP在饲料行业，尤其是水产行业具有潜在的应用前景。

关键词: 假单孢菌(Pseudomonas fluorescens), 植酸酶, 基因克隆与表达, 酶学性质

Abstract: A Pseudomonas fluorescens 206 strain producing HAP phytase was isolated from the bottom mud of artificial fish ponds in Jiaxin city. A novel HAP phytase encoding gene phyP was cloned by degenerate PCR and TAIL-PCR. The full length gene consist 1 284 bp and encodes 427 amino acids, including a putative signal peptide of 24 residues. The gene phyP was expressed in E.coli. The recombinant enzyme PhyP was purified to homogeneity by ammonium sulfalte precipitation and anion exchange chromatography. The optimal pH of recombinant PhyP was pH5.5, and the enzyme showed high stability at pH3.5-7.5. The optimal temperature of PhyP was found to be 45 ℃, and the enzyme had a higher activity at 25 ℃. These superior properties of PhyP make it advantageous for applying in feed industries, especially fisher industry.

Key words: Pseudomonas fluorescens, HAP phytase, Gene cloning and expression, Characteristics

李雅楠, 黄火清, 姚斌, 赵青, 刘昆, 马文康. 来源于假单孢菌206植酸酶基因的克隆、表达及酶学性质研究[J]. 生物技术通报, 2013, 0(8): 139-144.

Li Yanan, Huang Huoqing, Yao Bin, Zhao Qing, Liu Kun, Ma Wenkang. Gene Cloning，Expression and Characterization of an HAP Phytase from Pseudomonas fluorescens 206[J]. Biotechnology Bulletin, 2013, 0(8): 139-144.

参考文献

[1] Chen CC, Wu PH, Huang CT, et al. A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase[J]. Enz Microbial Technol, 2004, 35（4）：315-320.
[2] 姚斌, 范云六.植酸酶的分子生物学与基因工程[J].生物工程学报, 2000, 16（1）：1-5.
[3] Lei XG, Porres JM. Phytase enzymology, applications, and biotechnology[J]. Biotechnol Lett, 2003, 25（21）：1787-1794.
[4] Stahl CH, Roneker KR, Thornton JR, et al. A new phytase expressed in yeast effectively improves the bioavailability of phytate phosphorus to weanling pigs[J]. J Anim Sci, 2000, 78（3）：668-674.
[5] Augspurger NI, Webel DM, Lei XG, et al. Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs[J]. J Anim Sci, 2003, 81（2）：474-483.
[6] 罗琳, 吴秀峰, 薛敏, 等.中性植酸酶在豆粕型饲料中替代磷酸二氢钙对花鲈生长及磷代谢的影响[J].动物营养学报, 2007, 19（1）：33-39.
[7] Ravindran V, Cabahug S, Ravindran G, et al. Influence of microbial phytase on apparent ileal amino acid digestibility of feedstuffs for broilers[J]. Poult Sci, 1999, 78（5）：699-706.
[8] Cowieson AJ, Acamovic T, Bedford MR. The effects of phytase and phytic acid on the loss of endogenous amino acids and minerals from broiler chickens[J]. Br Poult Sci, 2004（45）：101-108.
[9] 贺建华.植酸磷和植酸酶研究进展[J].动物营养学报, 2005, 17（1）：1-6.
[10] Mullaney EJ, Ullah AH. The term phytase comprises several different classes of enzymes[J]. Biochem Biophys Res Commun, 2003, 312（1）：179-184.
[11] 白东清, 乔秀亭, 魏东, 等.植酸酶对鲤钙磷等营养物质利用率的影响[J].天津农学院学报, 2003, 10（1）：6-10.
[12] Sajjdi M, Carter CG. Dietary phytase supplementation and the utilization of phosphorus by Atlantic salmon fed a canola-meal-based diet[J]. Aquaculture, 2004, 240（1-4）：417-431.
[13] Howson SJ, Davis RP. Production of phytate hydrolyzing enzyme by some fungi[J]. Enzyme Microb Technol, 1983, 5（5）：377-382.
[14] Makarewicz O, Dubrac S, Msadek T, et al. Dual role of the PhoP-P response regulator：Bacillus amyloliquefaciens FZB45 phytase gene transcription is directed by positive and negative interactions with the phyC promoter[J]. J Bacteriol, 2006, 188（19）：6953-6965.
[15] Huang H, Shao N, Wang Y, et al. A novel beta-propeller phytase from Pedobacter nyackensis MJ11 CGMCC 2503 with potential as an aquatic feed additive[J]. Appl Microbiol Biotechnol, 2009, 83（2）：249-259.
[16] Liu YG, Whittier RF. Thermal asymmetric interlaced PCR：automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking[J]. Genomics, 1995, 25（3）：674-681.
[17] Huang H, Luo H, Yang P, et al. A novel phytase with preferable characteristics from Yersinia intermedia[J]. Biochem Biophys Res Commun, 2006, 350（4）：884-889.