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Journal of Zhejiang University SCIENCE A 2006 Vol.7 No.11 P.1948-1955


Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution

Author(s):  ZHANG Xiu-yan, RUAN Hui, MU Lin, HE Guo-qing, TANG Xing-jun, CHEN Qi-he

Affiliation(s):  Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China

Corresponding email(s):   gqhe@zju.edu.cn

Key Words:  Directed evolution, Error-prone PCR, DNA shuffling, &beta, -1, 3-1, 4-glucanase, Thermostability

ZHANG Xiu-yan, RUAN Hui, MU Lin, HE Guo-qing, TANG Xing-jun, CHEN Qi-he. Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution[J]. Journal of Zhejiang University Science A, 2006, 7(11): 1948-1955.

@article{title="Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution",
author="ZHANG Xiu-yan, RUAN Hui, MU Lin, HE Guo-qing, TANG Xing-jun, CHEN Qi-he",
journal="Journal of Zhejiang University Science A",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution
%A ZHANG Xiu-yan
%A MU Lin
%A HE Guo-qing
%A TANG Xing-jun
%A CHEN Qi-he
%J Journal of Zhejiang University SCIENCE A
%V 7
%N 11
%P 1948-1955
%@ 1673-565X
%D 2006
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.2006.A1948

T1 - Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution
A1 - ZHANG Xiu-yan
A1 - RUAN Hui
A1 - MU Lin
A1 - HE Guo-qing
A1 - TANG Xing-jun
A1 - CHEN Qi-he
J0 - Journal of Zhejiang University Science A
VL - 7
IS - 11
SP - 1948
EP - 1955
%@ 1673-565X
Y1 - 2006
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.2006.A1948

In order to improve the thermostability of &beta;-1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the &beta;-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGs1 and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5 °C for the wild-type enzyme to Tm=65.5 °C for the mutant EGs1 and 67.5 °C for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher (28%) for the former or much lower (21.6%) for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.

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


[1] Arnold, F.H., 2001. Combinatorial and computational challenges for biocatalyst design. Nature, 409(6817):253-257.

[2] Arnold, F.H., Volkov, A.A., 1999. Directed evolution of biocatalysts. Curr. Opin. Chem. Biol., 3(1):54-59.

[3] Babbitt, P.C., Gerlt, J.A., 1997. Understanding enzyme superfamilies: chemistry as the fundamental determinant in the evolution of new catalytic activities. J. Biol. Chem., 272(49):30591-30594.

[4] Cantwell, B.A., McConnell, D.J., 1983. Molecular cloning and expression of Bacillus subtilis β-glucanase gene in Escherichia coli. Gene, 23(2):211-219.

[5] DeSantis, G., Shang, X., Jones, J.B., 1999. Toward tailoring the specificity of the S1 pocket of subtilisin B. lentus: chemical modification of mutant enzymes as a strategy for removing specificity limitations. Biochemistry, 38(40):13391-13397.

[6] Edney, M.J., Marchylo, B.A., Macgregor, A.W., 1991. Structure of total barley-glucan. J. Inst. Brew., 97(1):39-44.

[7] Fincher, G.B., 1975. Morphology and chemical composition of barley endosperm cell walls. J. Inst. Brew., 81(2):116-122.

[8] Godfrey, T., Reinchelt, J., 1983. Industrial Enzymology. McMillan, London, p.466.

[9] Kurth, T., Grahn, S., Thormann, M., Ullmann, D., Hofmann, H.J., Jakubke, H.D., Hedstrom, L., 1998. Engineering the S1’ subsite of trypsin: design of a protease which cleaves between dibasic residues. Biochemistry, 37(33):11434-11440.

[10] Mouratou, B., Kasper, P., Gehring, H., Christen, P., 1999. Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase, an enzyme not found in Nature. J. Biol. Chem., 274(3):1320-1325.

[11] O’Brien, P.J., Herschlag, D., 1999. Catalytic promiscuity and the evolution of new enzymatic activities. Chem. Biol., 6(4):R91-R105.

[12] Planas, A., 2000. Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochimica et Biophysica Acta, 1543(2):361-382.

[13] Rohlin, L., Liao, J.C., 2001. Microbial pathway engineering for industrial processes: evolution, combinatorial biosynthesis and rational design. Curr. Opin. Microbiol., 4(3):330-335.

[14] Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual (2rd Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[15] Scott, R.W., 1972. The viscosity of worts in relation to their content of β-glucan. J. Inst. Brew., 78(2):179-186.

[16] Song, J.K., Rhee, J.S., 2000. Simultaneous enhancement of thermostability and catalytic activity of phospholipase A1by evolution molecular engineering. Applied and Environmental Microbiology, 66(3):890-894.

[17] Stemmer, W.P.C., 1994. DNA shuffling by random fragmentation and reassembly in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA, 91(22):10747-10751.

[18] Vieille, C., Zeikus, G.J., 2001. Hyperthermophilic enzyme: sources, uses, and molecular mechanisms for thermostability. Microbiology and Molecular Biology Reviews, 65(1):1-43.

[19] Wan, M.B., Twitchet, L.D., Eltis, A.G., Mauk, M., 1998. In vitro evolution of horse heart myoglobin to increase peroxidase activity. Proc. Natl. Acad. Sci. USA, 95(22):12825-12831.

[20] Woodward, J.R., Phillips, D.R., Fincher, G.B., 1983. Water soluble (1-3),(1-4)-β-D-glucans from barley (Hordeum vulgare) endosperm. I. Physicochemical properties. Carbohydr. Polym., 3(2):143-156.

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