海藻糖合酶的自诱导表达及其催化生产海藻糖的新工艺

doi:10.16085/j.issn.1000-6613.2017-1428

化工进展 ›› 2018, Vol. 37 ›› Issue (05): 1949-1955.DOI: 10.16085/j.issn.1000-6613.2017-1428

海藻糖合酶的自诱导表达及其催化生产海藻糖的新工艺

晏星辰^1,2, 赵倩如³, 王凯峰³, 郭玉欣³, 江凌², 黄和⁴

1 南京工业大学生物与制药工程学院, 江苏南京 210009;
2 南京工业大学食品与轻工学院, 江苏南京 210009;
3 南京工业大学2011学院, 江苏南京 210009;
4 南京工业大学药学院, 江苏南京 210009

收稿日期:2017-07-11 修回日期:2017-11-03 出版日期:2018-05-05 发布日期:2018-05-05
通讯作者: 江凌,研究员,研究方向为发酵工程和酶工程,
作者简介:晏星辰(1993-),男,硕士研究生。
基金资助:
国家自然科学基金联合基金（U1603112），生命有机化学国家重点实验室开放课题（SKLBNPC15429），国家重点研发计划（2017YFC1600404），江苏省环保厅项目（2016053）、江苏省十二批六大人才高峰（2015-JY-009）及江苏省先进生物制造创新中心项目。

Auto-induced expression of trehalose synthetase and novel process for catalytic production of trehalose

YAN Xingchen^1,2, ZHAO Qianru³, WANG Kaifeng³, GUO Yuxin³, JIANG Ling², HUANG He⁴

1 Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, Jiangsu, China;
2 College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, Jiangsu, China;
32011 College, Nanjing Tech University, Nanjing 211816, Jiangsu, China;
4 School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211816, Jiangsu, China

Received:2017-07-11 Revised:2017-11-03 Online:2018-05-05 Published:2018-05-05

摘要/Abstract

摘要： 海藻糖合成酶一步合成法是制备海藻糖最具工业化前景的方法之一。在前期研究基础上，本研究从发酵产酶和酶催化两方面进一步降低海藻糖生产成本，提高生产效率。首先，采用自诱导表达系统，在摇瓶体系中对含有海藻糖合成酶的大肠杆菌BL21（DE3）进行三阶段温度控制发酵，即以自诱导培养基发酵培养重组大肠杆菌，在37℃培养2.5h，25℃培养10.5h，30℃继续培养至发酵终点；此时，酶活达到1.42×10⁴U/mL，比自诱导恒温提高了57.4%。在此基础上，在5L反应器上进行三阶段温控发酵验证实验。通过连续发酵40h后OD达到31，细胞干重浓度达到15.32g/L，酶活达到最高值1.81×10⁴U/mL，比摇瓶发酵提高了28.6%。其次，开展全细胞催化生产海藻糖工艺优化，以5%甲苯处理45min的大肠杆菌细胞为研究对象，考察了pH、反应温度、底物浓度对全细胞催化合成海藻糖的影响以及全细胞催化的重复使用性能。结果发现：催化反应在pH为7.5、温度为30℃、麦芽糖初始浓度为350g/L时，海藻糖的得率达到74%以上；全细胞催化反应10个批次后，细胞仍保持对麦芽糖64.1%的转化率。本研究为海藻糖的工业化生产提供了一种经济、高效的工艺路线。

关键词: 海藻糖, 海藻糖合酶, 自诱导表达, 三阶段温控, 全细胞催化

Abstract: One-step catalysis by the trehalose synthase is one of the most promising methods for the trehalose production. Based on the previous researches,this study started with two aspects of enzyme expression and catalysis to reduce the cost and improve the productivity of trehalose production. Firstly,Escherichia coli BL21 (DE3)containing trehalose synthase was cultured using auto-induced expression system and three-stage temperature controlling strategy in shake flasks. The recombinant E. coli cells were sustainedly cultivated in 37℃ for 2.5h,25℃ for 10.5 h,and 30℃ till to the end of cultivation,respectively. The enzyme activity reached 1.42×10⁴U/mL,which was 57.4% higher than that obtained from the process of the constant temperature incubation. Furthermore,this cultivating strategy was conducted on a 5L bioreactor,where the fermentation lasted for 40h. At the end of fermentation,the OD value reached 31,which was equivalent to the cell dry weight concentration of 15.32g/L. Meanwhile,the enzyme activity reached 1.81×104 U/mL,which was 28.6% higher than the value obtained in shake flasks. Second,the recombinant E. coli cells treated with 5% toluene for 45min were used to optimize the process of whole cell production of trehalose,where the effects of pH,reaction temperature and substrate concentration on trehalose synthesis as well as the reuse efficiency of the whole-cell catalysis were investigated. It was found that the yield of trehalose reached more than 74% under the catalytic conditions of pH 7.5,30℃ and 350g/L of maltose,and remained as high as 64.1% at the 10th batch cycle. The results indicated that the new process could be a more economical and efficient process for the mass production of trehalose in the near future.

Key words: trehalose, trehalose synthase, auto-induced expression, three-stage temperature controlling strategy, whole-cell biocatalysis

中图分类号:

TQ464.1

晏星辰, 赵倩如, 王凯峰, 郭玉欣, 江凌, 黄和. 海藻糖合酶的自诱导表达及其催化生产海藻糖的新工艺[J]. 化工进展, 2018, 37(05): 1949-1955.

YAN Xingchen, ZHAO Qianru, WANG Kaifeng, GUO Yuxin, JIANG Ling, HUANG He. Auto-induced expression of trehalose synthetase and novel process for catalytic production of trehalose[J]. Chemical Industry and Engineering Progress, 2018, 37(05): 1949-1955.

参考文献

[1] EROGLU A,RUSSO M J,BIEGANSKI R,et al. Intracellular trehalose improves the survival of cryopreserved mammalian cells[J]. Nature Biotechnology,2000,18(2):163-167.
[2] ELBEIN A D,PAN Y T,PASTUSZAK I,et al. New insights on trehalose:a multifunctional molecule[J]. Glycobiology,2003,13(4):17-27.
[3] LIN F L,HALBEEK H V,BERTOZZI C R. Synthesis of mono-and dideoxygenated α,α-trehalose analogs[J]. Nitional Institutes of Health,2007,342(14):2014-2030.
[4] OHTAKE S,WANG Y J. Trehalose:current use and future applications[J]. Journal of Pharmaceutical Sciences,2011,100(6):2020-2053.
[5] MAYER C E,GEERLOF A,SCHEPERS A. Efficient expression and purification of tag-free Epstein-Barr virus EBNA1 protein in Escherichia coli by auto-induction[J]. Protein Expression and Purification,2012,86:7-11.
[6] GOUDARZI Z,SHOJAOSADATI S A,SAJEDI R H. A maghsoudi optimization of auto-induction conditions for the heterologous expression of a maltogenic amylase in Escherichia coli[J]. Applied Food Biotechnology,2016,3:105-113.
[7] CHEN W B,NIE Y,MU X Q,et al. Auto-induction-based rapid evaluation of extracellular enzyme expression from lac,operator-involved recombinant Escherichia coli[J]. Applied Biochemistry and Biotechnology,2014,174(7):2516-2526.
[8] 杨鑫,吴茜,崔怀言,等. 灰产色链霉菌海藻糖合成酶的自诱导表达及两阶段温度控制发酵的优化[J]. 食品与发酵工业,2015,41(5):14-18. YANG X,WU Q,CUI H Y,et al. Auto-induced expression of Streptomyces griseochromogenes trehalose synthase gene in Escherichia coli and optimization of its two-stage temperature controlling fermentation[J]. Food and Fermentation Industries,2015,41(5):14-18.
[9] ZHOU Y,YUAN Q P,GAO H L,et al. Production of trehalose by permeabilized Micrococcus QS412 cells[J]. Journal of Molecular Catalysis,2006,43:137-141.
[10] ZHENG Z,XU Y,SUN Y,et al. Biocatalytic production of trehalose from maltose by using whole cells of permeabilized recombinant Escherichia coli[J]. PLoS One,2015,10(10):e0140477.
[11] MOHAMED Y F,ABOUSHLEIB H M,KHALIL A M,et al. Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates[J]. Brazilian Journal of Microbiology,2016,47(2):381-388.
[12] STRAATHOF A J J,PANKE S,SCHMID A. The production of fine chemicals by biotransformations[J]. Current Opinionin Biotechnology,2002,13(6):548-556.
[13] SHAO M,ZHANG X,RAO Z,et al. Enhanced production of androst-1,4-diene-3,17-dione by Mycobacterium neoaurum JC-12 using three-stage fermentation strategy[J]. PLoS One,2015,10(9):e0137658.
[14] UHLIN B E,MOLIN S,GUSTAFEEON P,et al. Plasmids with temperature-dependent copy number for amplification of cloned genes and their products[J]. Gene,1979,6(2):91-106.
[15] ZHOU F,WANG C,WEI J. Simultaneous acetic acid separation and monosaccharide concentration by reverse osmosis[J]. Bioresource Technology,2013,131:349-356.
[16] ZHOU F,WANG C,WEI J. Repeated pH-stat fed-batch fermentation for rhamnolipid production with indigenous Pseudomonas aeruginosa S2[J]. Applied Microbiology and Biotechnology,2007,76(1):67-74.
[17] KUO C C,LIN C A,CHEN J Y,et al. Production of cyclodextrin glucanotransferase from an Alkalophilic Bacillus sp. by pH-stat fed-batch fermentation[J]. Biotechnology Letters,2009,31(11):1723-1727.
[18] NORD F F. Denaturation and inactivation of enzyme proteins[M]//Advances in enzymology and related areas of molecular biology, Wiley Online Library,2006:29-82.
[19] WANG J H,TSAI M Y,LEE G C,et al. Construction of a recombinant thermostable beta-amylase-trehalose synthase bifunctional enzyme for facilitating the conversion of starch to trehalose[J]. Journal of Agricultural and Food Chemistry,2007,55(4):1256-1263.
[20] LI Y,WANG Z,FENG Y,et al. Improving trehalose synthase activity by adding the C-terminal domain of trehalose synthase from Thermus thermophilus.[J]. Bioresource Technology,2017,245:1749-1756.
[21] LIANG J,HUANG R,HUANG Y,et al. Cloning,expression,properties,and functional amino acid residues of new trehalose synthase from Thermomonospora curvata,DSM 43183[J]. Journal of Molecular Catalysis B:Enzymatic,2013,90(355):26-32.
[22] COLLA E,PEREIRA A B,HERNALSTEENS S,et al. Optimization of trehalose production by rhodotorula dairenensis,following a sequential strategy of experimental design[J]. Food and Bioprocess Technology,2010,3(2):265-275.

海藻糖合酶的自诱导表达及其催化生产海藻糖的新工艺

Auto-induced expression of trehalose synthetase and novel process for catalytic production of trehalose

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 2

编辑推荐

Metrics

本文评价

[1]	纪淑兰, 李迅, 王飞. 丝瓜络固定化米根霉催化光皮树油制备生物柴油[J]. 化工进展, 2022, 41(10): 5381-5389.
[2]	秦晴，胡纯铿，洪美，高培培，顾中凤. 氮源影响酵母耐受超高浓度乙醇发酵胁迫条件 [J]. 化工进展, 2010, 29(9): 1719-.