Abstract
In order to improve the thermostability of β-1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-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 T m=62.5 °C for the wild-type enzyme to T m=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.
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References
Arnold, F.H., 2001. Combinatorial and computational challenges for biocatalyst design. Nature, 409(6817):253–257. [doi:10.1038/35051731]
Arnold, F.H., Volkov, A.A., 1999. Directed evolution of biocatalysts. Curr. Opin. Chem. Biol., 3(1):54–59. [doi:10.1016/S1367-5931(99)80010-6]
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. [doi:10.1074/jbc.272.49.30591]
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. [doi:10.1016/0378-1119(83)90053-7]
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. [doi:10.1021/bi990861o]
Edney, M.J., Marchylo, B.A., Macgregor, A.W., 1991. Structure of total barley-glucan. J. Inst. Brew., 97(1):39–44.
Fincher, G.B., 1975. Morphology and chemical composition of barley endosperm cell walls. J. Inst. Brew., 81(2):116–122.
Godfrey, T., Reinchelt, J., 1983. Industrial Enzymology. McMillan, London, p.466.
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. [doi:10.1021/bi980842z]
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. [doi:10.1074/jbc.274.3.1320]
O’Brien, P.J., Herschlag, D., 1999. Catalytic promiscuity and the evolution of new enzymatic activities. Chem. Biol., 6(4):R91–R105. [doi:10.1016/S1074-5521(99)80033-7]
Planas, A., 2000. Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochimica et Biophysica Acta, 1543(2):361–382.
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. [doi:10.1016/S1369-5274(00)00213-7]
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual (2rd Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Scott, R.W., 1972. The viscosity of worts in relation to their content of β-glucan. J. Inst. Brew., 78(2):179–186.
Song, J.K., Rhee, J.S., 2000. Simultaneous enhancement of thermostability and catalytic activity of phospholipase Alby evolution molecular engineering. Applied and Environmental Microbiology, 66(3):890–894. [doi:10.1128/AEM.66.3.890-894.2000]
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. [doi:10.1073/pnas.91.22.10747]
Vieille, C., Zeikus, G.J., 2001. Hyperthermophilic enzyme: sources, uses, and molecular mechanisms for thermostability. Microbiology and Molecular Biology Reviews, 65(1):1–43. [doi:10.1128/MMBR.65.1.1-43.2001]
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. [doi:10.1073/pnas.95.22.12825]
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. [doi:10.1016/0144-8617(83)90004-8]
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Project supported by the National Natural Science Foundation of China (No. 20276064) and Natural Science Foundation of Zhejiang Province (No. Z304076), China
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Zhang, Xy., Ruan, H., Mu, L. et al. Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution. J. Zhejiang Univ. - Sci. A 7, 1948–1955 (2006). https://doi.org/10.1631/jzus.2006.A1948
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DOI: https://doi.org/10.1631/jzus.2006.A1948