Constitutive Expression of Cyclodextrin Glycosyltransferase in <i>Pichia pastoris</i>

CHEN Long-jun; CHEN Ji-chen; LIN Xin-jian; CAI Hai-song

doi:10.19303/j.issn.1008-0384.2017.01.017

Volume 32 Issue 1

Mar. 2017

Turn off MathJax

Article Contents

Article Navigation > Fujian Journal of Agricultural Sciences > 2017 > 32(1): 82-86

CHEN Long-jun, CHEN Ji-chen, LIN Xin-jian, CAI Hai-song. Constitutive Expression of Cyclodextrin Glycosyltransferase in Pichia pastoris[J]. Fujian Journal of Agricultural Sciences, 2017, 32(1): 82-86. doi: 10.19303/j.issn.1008-0384.2017.01.017

Citation:

CHEN Long-jun, CHEN Ji-chen, LIN Xin-jian, CAI Hai-song. Constitutive Expression of Cyclodextrin Glycosyltransferase in Pichia pastoris[J]. Fujian Journal of Agricultural Sciences, 2017, 32(1): 82-86. doi: 10.19303/j.issn.1008-0384.2017.01.017

Citation:

PDF( 1345 KB)

Constitutive Expression of Cyclodextrin Glycosyltransferase in Pichia pastoris

doi: 10.19303/j.issn.1008-0384.2017.01.017

Soil and Fertilizer Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China

Received Date: 2016-04-28
Rev Recd Date: 2016-06-29
Publish Date: 2017-01-01

Abstract

Abstract

This study aimed to obtain a highly efficient expression of cyclodextrin glycosyltransferase (CGTase) in Pichia pastoris. The optimized CGT2 was cloned into a yeast constitutive expression vector, pGATZαA.The recombinant plasmid pGAPZαA-CGT2 was then transformed into P. pastoris by electroporation to construct an engineered strain, X33/pGAPZαA-CGT2.After cultivating for 120 h in a shaking flask, CGT2 with an activity of 0.21 U·mL^-1 was obtained. Experiments were conducted to further optimize the fermentation conditions. As a result, the greatest activity of 1.26 U·mL^-1, achieving a 1.7-fold improvement, for the enzyme was reached by induction for 120 h at pH 6.5 and 28℃ with a constant shaking at 200 r·min^-1 and replenishing with 2.5% glycerol every 24 h in the flask.
- cyclodextrin glycosyltransferase,
- Pichia pastoris,
- constitutive expression,
- fermentation

FullText(HTML)

References(12)

References

[1]	LI Z F, WANG M, WANG F, et al.Gamma-cyelodextfin:a review on enzymatic production and applications[J].Apply Microbiol Biotechnol, 2007, 77(2):245-255. doi: 10.1007/s00253-007-1166-7
[2]	LI Z, LI B, GU Z, et al.Extracellular expression and biochemical Characterization of alpha-cyclodextrin glycosyl-transferase from Paenibacillus macerans[J].Carbohydrate research, 2010, 345(7):886-892. doi: 10.1016/j.carres.2010.02.002
[3]	金征宇, 徐学明, 陈寒青, 等.环糊精化学-制备与应用[M].北京:化学工业出版, 2005:1-35.
[4]	李兆丰. 软化类芽孢杆菌α-环糊精葡萄糖基转移酶在大肠杆菌中的表达及其产物特异性分析[D]. 无锡: 江南大学, 2009.
[5]	PENKA P, ALEXANDRA T, KALOYAN P. Sequence analysis, cloning and extracellular expression of cyclodextrin glucanotransferase gene from the alkaliphilic Bacillus pseudalcaliphilus 8SB in Escherichia coli[J].Process Biochemistry, 2012, 47(12):2139-2145. doi: 10.1016/j.procbio.2012.08.001
[6]	IU H, I J H, U G C, et al. Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization[J]. Journal of Industrial Microbiology & Biotechnology, 2012:1841-1849. https://www.researchgate.net/publication/230685542_Enhanced_production_of_a-cyclodextrin_glycosyltransferase_in_Escherichia_coli_by_systematic_codon_usage_optimization
[7]	JARUNEE K, WANIDA P, VICHIEN R, et al. Expression and characterization of a fusion protein-containing cyclodextrin glycosyltransferase from Paenibacillus sp. A11[J].Journal of Basic Microbiology, 2010, 50(5):427-435. doi: 10.1002/jobm.200900039
[8]	NAM S W, PARK H Y, KIM J H, et al. Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Sacchoromyces cerevisiae[J].Biotechnol Lett, 2001, 23(9):727-730. doi: 10.1023/A:1010303019603
[9]	张佳瑜, 吴丹, 李兆丰, 等.来源于软化芽孢杆菌的环糊精葡萄糖基转移酶在毕赤酵母和枯草杆菌中的表达[J].生物工程学报, 2009, 25(12):1948-1954. http://www.cnki.com.cn/Article/CJFDTOTAL-SHWU200912028.htm
[10]	ZHAO W, WANG J, DENG R, et al. Scale-up fermentation of recombinant Candida rugosa lipase expressed in Pichia pastoris using the GAP promoter[J]. Journal of Industrial Microbiology and Biotechnolog, 2008, 35(3):189-195. doi: 10.1007/s10295-007-0283-8
[11]	曹东艳, 柳倩, 贺晓云, 等.源于GAP启动子的毕赤酵母组成型表达系统的研究进展[J].食品安全质量检测学报, 2013, 4(4):1217-1221. http://www.cnki.com.cn/Article/CJFDTOTAL-SPAJ201304054.htm
[12]	LEJEUNE A, SAKAGUCHI K, IMANAKA T. A spectrophotometric assay for the cyclization activity of cyclomaltohexaose (alpha-cyclodextrin) glucanotransferase[J]. Analytical biochemistr, 1989, 181(1):6-11. doi: 10.1016/0003-2697(89)90385-0