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Applied Microbiology and Biotechnology

, Volume 77, Issue 3, pp 569–578 | Cite as

Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity

  • Ai-Sheng Xiong
  • Ri-He Peng
  • Jing Zhuang
  • Xian Li
  • Yong Xue
  • Jin-Ge Liu
  • Feng Gao
  • Bin Cai
  • Jian-Min Chen
  • Quan-Hong Yao
Biotechnologically Relevant Enzymes and Proteins

Abstract

We performed directed evolution on a chemically synthesized 1,533-bp recombinant beta-galactosidase gene from Pyrococcus woesei. More than 200,000 variant colonies in each round of directed evolution were screened using the pYPX251 vector and host strain Rosetta-Blue (DE3). One shifted beta-galactosidase to beta-glucuronidase mutant, named YG6762, was obtained after four rounds of directed evolution and screening. This mutant had eight mutated amino acid residues. T29A, V213I, L217M, N277H, I387V, R491C, and N496D were key mutations for high beta-glucuronidase activity, while E414D was not essential because the mutation did not lead to a change in beta-glucuronidase activity. The amino acid site 277 was the most essential because mutating H back to N resulted in a 50% decrease in beta-glucuronidase activity at 37°C. We also demonstrated that amino acid 277 was the most essential site, as the mutation from N to H resulted in a 1.5-fold increase in beta-glucuronidase activity at 37°C. Although most single amino acid changes lead to less than a 20% increase in beta-glucuronidase activity, the YG6762 variant, which was mutated at all eight amino acid sites, had a beta-glucuronidase activity that was about five and seven times greater than the wild-type enzyme at 37 and 25°C, respectively.

Keywords

Beta-galactosidase Beta-glucuronidase Pyrococcus woesei Directed evolution Enzyme properties Structure–function analysis 

Notes

Acknowledgment

This research was supported by the Shanghai Rising-Star Program (05QMX1445), the Program of Shanghai Subject Chief Scientist (06XD14017), the National and Shanghai Natural Science Foundation (30471258, 04ZR14116), the Shanghai Project for ISTC (055407068), and NAFC Program (2006GB2C000086).

Supplementary material

253_2007_1182_MOESM1_ESM.doc (64 kb)
Table 1 Primers for enzymatic synthesis of the hlacz-sh gene using the PTDS method. Red: forward primer; Blue: reverse primer. Shadow: Bam HI and Sac I sites (DOC 64 kb).
253_2007_1182_MOESM2_ESM.doc (27 kb)
Table 2 Primers used for DNA shuffling and sequencing in this study (DOC 27 kb).
253_2007_1182_MOESM3_ESM.doc (40 kb)
Fig. 1 Comparison of nucleotide sequences between the hlacz-sh gene (GenBank accession no. EF090269) and the β-galactosidases gene from P. woesei (GenBank accession no. AF043283) (DOC 40.5 kb).
253_2007_1182_MOESM4_ESM.doc (330 kb)
Fig. 2 The colonies containing the mutated yg6762 gene (YG6762-left) exhibited more β-glucuronidase activity than the colonies harbouring the wild-type synthesized 1,533-bp recombinant β-galactosidases gene from P. woesei (YH4502-right). After incubation of 1.5 hours, the colonies hosting wild-type hlacz-sh gene became blue because of the background β-glucuronidase of the host E. coli strain DH5α. (a). Nitrocellulose filter was incubated in X-Gal at 37°C for 0 hour. (b). Nitrocellulose filter was incubated in X-GlcA at 37°C for 30 min. (c). Nitrocellulose filter was incubated in X-GlcA at 37°C for 1 hours. (d). Nitrocellulose filter was incubated in X-GlcA at 37°C for 2 hours. (e). Nitrocellulose filter was incubated in X-GlcA at 37°C for 4 hours (DOC 330 kb).
253_2007_1182_MOESM5_ESM.doc (108 kb)
Fig. 3 Purified wild-type β-galactosidase (YH4502-WT) and the variant that showed high β-glucuronidase activity (YG6762). Lane M: protein marker; Lane WT: Purified wild-type β-galactosidase (YH4502-WT); Lane YG6762: Purified YG6762 (DOC 108 kb).
253_2007_1182_MOESM6_ESM.doc (26 kb)
Fig. 4 The specific activity of β-galactosidase and β-glucuronidase from wild-type (YH4502) and variant (YG6762) enzymes at 25°C, 37°C, 90°C and 100°C. (a), β-glucuronidase activity. (b), β-galactosidase activity (DOC 26.5 kb).

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Ai-Sheng Xiong
    • 1
  • Ri-He Peng
    • 1
  • Jing Zhuang
    • 1
  • Xian Li
    • 1
  • Yong Xue
    • 1
  • Jin-Ge Liu
    • 1
  • Feng Gao
    • 1
  • Bin Cai
    • 1
  • Jian-Min Chen
    • 2
  • Quan-Hong Yao
    • 1
  1. 1.Biotechnology Research InstituteShanghai Academy of Agricultural SciencesShanghaiChina
  2. 2.College of Bioscience and BiotechnologyYangzhou UniversityYangzhouChina

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