Full Text:   <2602>

CLC number: Q26

On-line Access: 2011-07-04

Received: 2010-07-06

Revision Accepted: 2011-01-10

Crosschecked: 2011-06-16

Cited: 7

Clicked: 5274

Citations:  Bibtex RefMan EndNote GB/T7714

-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2011 Vol.12 No.7 P.545-551


Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Author(s):  Mei-ling Chen, Qin Guo, Rui-zhi Wang, Juan Xu, Chen-wei Zhou, Hui Ruan, Guo-qing He

Affiliation(s):  Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China, School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

Corresponding email(s):   ruanhui98@yahoo.com.cn

Key Words:  Rhizopus oryzae lipase (ROL), Yeast surface display, Codon optimization, Whole-cell biocatalyst

Mei-ling Chen, Qin Guo, Rui-zhi Wang, Juan Xu, Chen-wei Zhou, Hui Ruan, Guo-qing He. Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity[J]. Journal of Zhejiang University Science B, 2011, 12(7): 545-551.

@article{title="Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity",
author="Mei-ling Chen, Qin Guo, Rui-zhi Wang, Juan Xu, Chen-wei Zhou, Hui Ruan, Guo-qing He",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity
%A Mei-ling Chen
%A Qin Guo
%A Rui-zhi Wang
%A Juan Xu
%A Chen-wei Zhou
%A Hui Ruan
%A Guo-qing He
%J Journal of Zhejiang University SCIENCE B
%V 12
%N 7
%P 545-551
%@ 1673-1581
%D 2011
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1000258

T1 - Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity
A1 - Mei-ling Chen
A1 - Qin Guo
A1 - Rui-zhi Wang
A1 - Juan Xu
A1 - Chen-wei Zhou
A1 - Hui Ruan
A1 - Guo-qing He
J0 - Journal of Zhejiang University Science B
VL - 12
IS - 7
SP - 545
EP - 551
%@ 1673-1581
Y1 - 2011
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1000258

Surface display is effectively utilized to construct a whole-cell biocatalyst. codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1]Andersson, S.G.E., Kurland, C.G., 1990. Codon preferences in free-living microorganisms. Microbiol. Rev., 54(2):198-210.

[2]Bucarey, S.A., Noriega, J., Reyes, P., Tapia, C., Saenz, L., Zuniga, A., Tobar, J.A., 2009. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. Vaccine, 27(42):5781-5790.

[3]Das, S., Roymondal, U., Sahoo, S., 2009. Analyzing gene expression from relative codon usage bias in yeast genome: a statistical significance and biological relevance. Gene, 443(1-2):121-131.

[4]Esteban, L., Munio, M.D., Robles, A., Hita, E., Jimenez, M.J., Gonzalez, P.A., Camacho, B., Molina, E., 2009. Synthesis of 2-monoacylglycerols (2-MAG) by enzymatic alcoholysis of fish oils using different reactor types. Biochem. Eng. J., 44(2-3):271-279.

[5]Guldener, U., Heck, S., Fiedler, T., Beinhauer, J., Hegemann, J.H., 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res., 24(13):2519-2524.

[6]Guo, Q., Zhang, W., Ma, L.L., Chen, Q.H., Chen, J.C., Zhang, H.B., Ruan, H., He, G.Q., 2010. A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 11(1):41-51.

[7]Hama, S., Yamaji, H., Kaieda, M., Oda, M., Kondo, A., Fukuda, H., 2004. Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production. Biochem. Eng. J., 21(2):155-160.

[8]Hasan, F., Shah, A.A., Hameed, A., 2006. Industrial applications of microbial lipases. Enzyme Microb. Technol., 39(2):235-251.

[9]Ikemura, T., 1985. Codon usage and transfer-RNA content in unicellular and multicellular organisms. Mol. Biol. Evol., 2(1):13-34.

[10]Kaewthong, W., Sirisansaneeyakul, S., Prasertsan, P., H-Kittikun., A., 2005. Continuous production of monoacylglycerols by glycerolysis of palm olein with immobilized lipase. Process Biochem., 40(5):1525-1530.

[11]Kato, M., Fuchimoto, J., Tanino, T., Kondo, A., Fukuda, H., Ueda, M., 2007. Preparation of a whole-cell biocatalyst of mutated Candida antaretica lipase B (mCALB) by a yeast molecular display system and its practical properties. Appl. Microbiol. Biotechnol., 75(3):549-555.

[12]Klibanov, A.M., 2001. Improving enzymes by using them in organic solvents. Nature, 409(6817):241-246.

[13]Kondo, A., Ueda, M., 2004. Yeast cell-surface display—applications of molecular display. Appl. Microbiol. Biotechnol., 64(1):28-40.

[14]Kristensen, J.B., Xu, X.B., Mu, H.L., 2005. Diacylglycerol synthesis by enzymatic glycerolysis: screening of commercially available lipases. J. Am. Oil Chem. Soc., 82(5):329-334.

[15]Kurland, C.G., 1991. Codon bias and gene-expression. FEBS Lett., 285(2):165-169.

[16]Lithwick, G., Margalit, H., 2003. Hierarchy of sequence-dependent features associated with prokaryotic translation. Genome Res., 13(12):2665-2673.

[17]Lo, S.K., Baharin, B.S., Tan, C.P., Lai, O.M., 2004. Lipase-catalysed production and chemical composition of diacylglycerols from soybean oil deodoriser distillate. Eur. J. Lipid Sci. Technol., 106(4):218-224.

[18]Matsumoto, T., Takahashi, S., Kaieda, M., Ueda, M., Tanaka, A., Fukuda, H., Kondo, A., 2001. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production. Appl. Microbiol. Biotechnol., 57(4):515-520.

[19]Murai, T., Ueda, M., Shibasaki, Y., Kamasawa, N., Osumi, M., Imanaka, T., Tanaka, A., 1999. Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface. Appl. Microbiol. Biotechnol., 51(1):65-70.

[20]Prim, N., Blanco, A., Martinez, J., Pastor, F.I.J., Diaz, P., 2000. estA, a gene coding for a cell-bound esterase from Paenibacillus sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases. Res. Microbiol., 151(4):303-312.

[21]Proshkin, S., Rahmouni, A.R., Mironov, A., Nudler, E., 2010. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science, 328(5977):504-508.

[22]Shibasaki, S., Maema, H., Ueda, M., 2009. Molecular display technology using yeast-arming technology. Anal. Sci., 25(1):41-49.

[23]Sinclair, G., Choy, F.Y.M., 2002. Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris. Protein Expr. Purif., 26(1):96-105.

[24]Tamalampudi, S., Talukder, M.R., Hama, S., Numata, T., Kondo, A., Fukuda, H., 2008. Enzymatic production of biodiesel from Jatropha oil: a comparative study of immobilized-whole cell and commercial lipases as a biocatalyst. Biochem. Eng. J., 39(1):185-189.

[25]Thompson, J.R., Register, E., Curotto, J., Kurtz, M., Kelly, R., 1998. An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast, 14(6):565-571.

[26]Washida, M., Takahashi, S., Ueda, M., Tanaka, A., 2001. Spacer-mediated display of active lipase on the yeast cell surface. Appl. Microbiol. Biotechnol., 56(5-6):681-686.

[27]Zaks, A., Klibanov, A.M., 1988. Enzymatic catalysis in nonaqueous solvents. J. Biol. Chem., 263(7):3194-3201.

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2024 Journal of Zhejiang University-SCIENCE