High Level Expression of a Recombinant Phytase appA -2QN in Pichia pastoris

doi:10.7668/hbnxb.2015.02.011

Abstract

Abstract: The gene appA for phytase from Escherchia coli was modified by means of site directed mutagenesis to improve its thermal stability.Using yeast expression system the mutant phytase appA -2QN produced in shaking-flask culture showed an increased thermal stability.To increase its expression level and lower production costs, an approach of the high cell-density fermentation of recombinant yeast GS115/ appA -2QN was carried out in this study.During fermentation the concentration of glycerin was controlled to promote the cell growth and achieve the high cell-density fermentation.The method of chromotropic acid spectrophotography was used to determine the concentration of methanol during fermentation.The results showed that the OD_600nm of the cell reached 313 after 147 h fermentation in a 5 L fermentor.After inducing of 102 h with methanol at a concentration of 2.5%, the expression level of phytase reached 7.06 g/L.The phytase activity was 2.03×10⁵ U/mL.These results indicated that the cell density and the expression level of target protein were significantly improved by high density fermentation.The recombinant yeast strain has favorable expression stability.

Key words: Recombinant phytase, Pichia pastoris, High cell-density fermentation, High level expression

CLC Number:

Q786

SONG Ma-li, WANG Xi, DU Jun, ZHAO Feng-mei, LIANG Ai-hua. High Level Expression of a Recombinant Phytase appA -2QN in Pichia pastoris[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(2): 55-58. doi: 10.7668/hbnxb.2015.02.011.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2015.02.011

http://www.hbnxb.net/EN/Y2015/V30/I2/55

References

[1] Mullaney E J,Daly C B,Ullah A H.Advances in phytase research[J].Advances in Applied Microbiology,2000,47:157-199.
[2] 王红艳,王云飞,王申涛.植酸酶的研究进展及应用[J].中国酿造,2010(4):24-25.
[3] 王郡美,宋维平,张莉.宇佐美曲霉植酸酶 phyA 基因的克隆[J].华北农学报,2006,21(3):5-8.
[4] 魏凤仙,白献晓,李绍钰,等.低磷加植酸酶日粮不同钙磷比对生长猪生产性能和血清学指标的影响[J].华北农学报,2007,22(5):47-50.
[5] Yao M Z,Zhang Y H,Lu W L,et al.Phytases:crystal structures,protein engineering and potential biotechnological applications[J].Journal of Applied Microbiology,2012,112(1):1-14.
[6] 谷维娜,杨培龙,王亚茹,等. Aspergillus fumigatus 来源植酸酶的Q23L和Q23LG272E突变研究[J].生物工程学报,2007,23(2):273-277.
[7] 胡骁飞,魏凤仙,李青梅,等.植酸酶单克隆抗体的制备及鉴定[J].华北农学报,2012,27(5):44-48.
[8] Sreekrishna K,Brankamp R G,Kropp K E,et al.Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris[J].Gene,1997,190(1,SI):55-62.
[9] 张梅申,沈爱光,熊晓辉,等.植酸酶产生菌株的液体深层发酵条件研究[J].华北农学报,1999,14(4):63-67.
[10] Xiong A S,Yao Q H,Peng R H,et al.High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris [J].Applied Microbiology and Biotechnology,2006,72(5):1039-1047.
[11] Yao M Z,Wang X,Wang W,et al.Improving the thermostability of Escherichia coli phytase,appA,by enhancement of glycosylation[J].Biotechnology Letters,2013,35(10):1669-1676.
[12] Clare J J,Rayment F B,Ballantine S P,et al.High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene[J].Biotechnology,1991,9(5):455-460.
[13] 姚明泽.植酸酶appA 的定向改造及Phy(ycE)的酶学性质研究[D].太原:山西大学,2013.
[14] 余波,戴小峰,张社,等.变色酸分光光度法测定毕赤酵母发酵液中的甲醇含量[J].上海大学学报:自然科学版,2006,12(2):196-199.
[15] 张耀华,姚明泽,卢文亮,等.大肠杆菌植酸酶在毕赤酵母中的表达与纯化[J].应用与环境生物学报,2012(4):678-681.
[16] Xiong A S,Yao Q H,Peng R H,et al.Isolation,characterization,and molecular cloning of the cDNA encoding a novel phytase from Aspergillus Niger 113 and high expression in Pichia pastoris [J].Journal of Biochemistry and Molecular Biology,2004,37(3):282-291.
[17] Stahl C H,Wilson D B,Lei X G.Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris [J].Biotechnology Letters,2003,25(10):827-831.
[18] 孙子羽,迟乃玉,董硕,等.低温植酸酶菌株Y1的发酵条件研究[J].生物技术,2010,20(5):72-75.
[19] 黄光瑞,田丽春,黄生平,等.密码子优化的植酸酶基因 appA-P 在毕赤酵母中的高效表达[J].湖北大学学报:自然科学版,2007,29(3):290-293.
[20] 王亚茹,姚斌,罗会颖,等.来源于 Selenomonas ruminantium 的高比活植酸酶基因在毕赤酵母中的高效表达[J].中国农业科学,2004,37(5):762-768.
[21] Khatri N K,Hoffmann F.Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastori s[J].Biotechnology and Bioengineering,2006,93(5):871-879.
[22] Mayson B E,Kilburn D G,Zamost B L,et al.Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica [J].Biotechnology and Bioengineering,2003,81(3):291-298.

Please choose a citation manager

Content to export