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 YaoEmail author
Biotechnologically Relevant Enzymes and Proteins


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.


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



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).


  1. Alexeeva M, Carr R, Turner NJ (2003) Directed evolution of enzymes: new biocatalysts for asymmetric synthesis. Org Biomol Chem 1:4133–4137CrossRefGoogle Scholar
  2. Castle LA, Siehl DL, Gorton R, Patten PA, Chen YH, Bertain S, Cho HJ, Duck N, Wong J, Liu D, Lassner MW (2004) Discovery and directed evolution of a glyphosate tolerance gene. Science 304:1151–1154CrossRefGoogle Scholar
  3. Coker JA, Sheridan PP, Loveland-Curtze J, Gutshall KR, Auman AJ, Brenchley JE (2003) Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic Arthrobacter isolate. J Bacteriol 185:5473–5482CrossRefGoogle Scholar
  4. Coombs JM, Brenchley JE (1999) Biochemical and phylogenetic analyses of a cold-active beta-galactosidase from the lactic acid bacterium Carnobacterium piscicola BA. Appl Environ Microbiol 65:5443–5450Google Scholar
  5. Crameri A, Whitehorn EA, Tate E, Stemmer WP (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14:315–319CrossRefGoogle Scholar
  6. Dabrowski S, Maciunska J, Synowiecki J (1998) Cloning and nucleotide sequence of the thermostable β-galactosidase gene from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme. Mol Biotechnol 10:217–222CrossRefGoogle Scholar
  7. Dabrowski S, Sobiewska G, Maciunska J, Synowiecki J, Kur J (2000) Cloning, expression, and purification of the His(6)-tagged thermostable beta-galactosidase from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme. Protein Expr Purif 19:107–112CrossRefGoogle Scholar
  8. Demir V, Dincturk HB (2006) Semi-anaerobic growth conditions are favoured by some Escherichia coli strains during heterologous expression of some archaeal proteins. Mol Biol Rep 33:59–63CrossRefGoogle Scholar
  9. Flores H, Ellington AD (2002) Increasing the thermal stability of an oligomeric protein, beta-glucuronidase. J Mol Biol 315:325–337CrossRefGoogle Scholar
  10. Fradkov AF, Chen Y, Ding L, Barsova EV, Matz MV, Lukyanov SA (2000) Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence. FEBS Lett 479:127–130CrossRefGoogle Scholar
  11. Geddie ML, Matsumura I (2004) Rapid evolution of beta-glucuronidase specificity by saturation mutagenesis of an active site loop. J Biol Chem 279:26462–26468CrossRefGoogle Scholar
  12. Gilissen LJ, Metz PL, Stiekema WJ, Nap JP (1998) Biosafety of E. coli beta-glucuronidase (GUS) in plants. Transgenic Res 7:157–163CrossRefGoogle Scholar
  13. Helmer G, Casadaban M, Bevan M, Kayes L, Chilton ML (1984) A new chimeric gene as a marker for plant transformation: the expression of Escherichia coli β-galactosidase in sunflower and tobacco cells. Biotechnology 2:520–527CrossRefGoogle Scholar
  14. Hibbert EG, Baganz F, Hailes HC, Ward JM, Lye GJ, Woodley JM, Dalby PA (2005) Directed evolution of biocatalytic processes. Biomol Eng 22:11–19CrossRefGoogle Scholar
  15. Holmes ML, Scopes RK, Moritz RL, Simpson RJ, Englert C, Pfeifer F, Dyall-Smith ML (1997) Purification and analysis of an extremely halophilic β-galactosidase from Haloferax alicantei. Biochim Biophys Acta 1337:276–286Google Scholar
  16. Hu C, Chee PP, Chee KPP, Chesney RH, Zhou JH, Miller PD, O’brien WT (1990) Intrinsic GUS-like activities in seed plants. Plant Cell Rep 9:1–5CrossRefGoogle Scholar
  17. Jacobson RH, Zhang XJ, DuBose RF, Matthews BW (1994) Three-dimensional structure of beta-galactosidase from E. coli. Nature 369:761–766CrossRefGoogle Scholar
  18. Jefferson RA, Kavanaugh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker for higher plants. EMBO J 6:3901–3907Google Scholar
  19. Kosugi S, Ohashi Y, Nakajima K, Arai Y (1990) An improved assay for β-glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenous β-glucuronidase activity. Plant Sci 70:133–140CrossRefGoogle Scholar
  20. Lassner M, Bedbrook J (2001) Directed molecular evolution in plant improvement. Curr Opin Plant Biol 4:152–156CrossRefGoogle Scholar
  21. Locher CP, Soong NW, Whalen RG, Punnonen J (2004) Development of novel vaccines using DNA shuffling and screening strategies. Curr Opin Mol Ther 6:34–39Google Scholar
  22. Locher CP, Paidhungat M, Whalen RG, Punnonen J (2005) DNA shuffling and screening strategies for improving vaccine efficacy. DNA Cell Biol 24:256–263CrossRefGoogle Scholar
  23. Matsumura I, Wallingford JB, Surana NK, Vize PD, Ellington AD (1999) Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase. Nat Biotechnol 17:696–701CrossRefGoogle Scholar
  24. Matthews BW (2005) The structure of E. coli beta-galactosidase. C R Biol 328:549–556CrossRefGoogle Scholar
  25. Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 107:193–232CrossRefGoogle Scholar
  26. Morley KL, Kazlauskas RJ (2005) Improving enzyme properties: when are closer mutations better. Trends Biotechnol 23:231–237CrossRefGoogle Scholar
  27. Murray EE, Lotzer J, Eberle M (1989) Codon usage in plant genes. Nucleic Acids Res 17:477–498CrossRefGoogle Scholar
  28. Nam SH, Oh KH, Kim GJ, Kim HS (2003) Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution. Protein Eng 16:1099–1105CrossRefGoogle Scholar
  29. Ow DW, Wood KV, DeLuca M, Wet JRD, Helinski DR, Howell SH (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856–859CrossRefGoogle Scholar
  30. Panesar PS, Panesar R, Singh RS, Kennedy JF, Kumar H (2006) Microbial production, immobilization and applications of β-d-galactosidase. J Chem Technol Biotechnol 81:530–543CrossRefGoogle Scholar
  31. Parikh MR, Matsumura I (2005) Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase. J Mol Biol 352:621–628CrossRefGoogle Scholar
  32. Rogers HJ, Maund SL, Johnson LH (2001) A beta-galactosidase-like gene is expressed during tobacco pollen development. J Exp Bot 52:67–75CrossRefGoogle Scholar
  33. Rowe LA, Geddie ML, Alexander OB, Matsumura I (2003) A comparison of directed evolution approaches using the beta-glucuronidase model system. J Mol Biol 332:851–860CrossRefGoogle Scholar
  34. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory mannual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  35. Sharp PM, Tuohy TMF, Mosurski KR (1986) Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expression genes. Nucleic Acids Res 14:5125–5143CrossRefGoogle Scholar
  36. Sheridan PP, Brenchley JE (2000) Characterization of a salt-tolerant family 42 beta-galactosidase from a psychrophilic Antarctic Planococcus isolate. Appl Environ Microbiol 66:2438–2444CrossRefGoogle Scholar
  37. Shipkowski S, Brenchley JE (2005) Characterization of an unusual cold-active beta-glucosidase belonging to family 3 of the glycoside hydrolases from the psychrophilic isolate Paenibacillus sp. strain C7. Appl Environ Microbiol 71:4225–4232CrossRefGoogle Scholar
  38. Simmonds J, Cass L, Routly E, Hubbard K, Donaldson P, Bancroft B, Davidson A, Hubbard S, Simmonds D (2004) Oxalate oxidase: a novel reporter gene for monocot and dicot transformations. Mol Breeding 13:79–91CrossRefGoogle Scholar
  39. Smith DL, Gross KC (2000) A family of at least seven beta-galactosidase genes is expressed during tomato fruit development. Plant Physiol 123:1173–1183CrossRefGoogle Scholar
  40. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefGoogle Scholar
  41. Smith HO, Hutchison 3rd CA, Pfannkoch C, Venter JC (2003) Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci U S A 100:15440–15445CrossRefGoogle Scholar
  42. Stefan A, Radeghieri A, Gonzalez A, Hochkoeppler A (2001) Directed evolution of beta-galactosidase from Escherichia coli by mutator strains defective in the 3′–>5′ exonuclease activity of DNA polymerase III. FEBS Lett 493:139–143CrossRefGoogle Scholar
  43. Stewart CN (2001) The utility of green fluorescent protein in transgenic plants. Plant Cell Rep 20:376–382CrossRefGoogle Scholar
  44. Trimbur DE, Gutshall KR, Prema P, Brenchley JE (1994) Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase. Appl Environ Microbiol 60:4544–4552Google Scholar
  45. Xiong AS, Yao QH, Peng RH, Li X, Fan HQ, Guo MJ, Zhang SL (2004a) Isolation, characterization, molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastoris. J Biochem Mol Biol 37:282–291Google Scholar
  46. Xiong AS, Yao QH, Peng RH, Li X, Fan HQ, Cheng ZM, Li Y (2004b) A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res 32:e98CrossRefGoogle Scholar
  47. Xiong AS, Yao QH, Peng RH, Han PL, Cheng ZM, Li Y (2005) High level expression of a recombinant acid phytase gene in Pichia pastoris. J Appl Microbiol 98:418–428CrossRefGoogle Scholar
  48. Xiong AS, Yao QH, Peng RH, Zhang Z, Xu F, Liu JG, Han PL, Chen JM (2006a) High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris. Appl Microbiol Biotechnol 72:1039–1047CrossRefGoogle Scholar
  49. Xiong AS, Yao QH, Peng RH, Duan H, Li X, Fan HQ, Cheng ZM, Li Y (2006b) PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1:791–797CrossRefGoogle Scholar
  50. Xiong AS, Peng RH, Zhuang J, Liu JG, Xu F, Cai B, Guo ZK, Qiao YS, Chen JM, Zhang Z, Yao QH (2007a) Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase. J Biochem Mol Biol 40:419–425Google Scholar
  51. Xiong AS, Peng RH, Liu JG, Zhuang J, Qiao YS, Xu F, Cai B, Zhang Z, Chen JM, Yao QH (2007b) High efficiency and throughput system in directed evolution in vitro of reporter gene. Appl Microbiol Biotechnol 74:160–168CrossRefGoogle Scholar
  52. Xiong AS, Peng RH, Cheng ZM, Li Y, Liu JG, Zhuang J, Feng G, Xu F, Qiao YS, Zhang Z, Chen JM, Yao QH (2007c) Concurrent mutations in six amino acids in β-glucuronidase improves its thermostability. Protein Eng Des Sel 20:319–325CrossRefGoogle Scholar
  53. Zhang JH, Dawes G, Stemmer WP (1997) Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc Natl Acad Sci U S A 94:4504–4509CrossRefGoogle Scholar
  54. Zillig W, Holz I, Klenk HP, Trent J, Wunderl S, Janekovic D, Imsel E, Haas B (1987) Pyrococcus woesei, sp. nov., an ultrathermophilic marine archaebacterium, representing a novel order. Thermococcales Syst Appl Microbiol 9:62–70Google Scholar

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
    Email author
  1. 1.Biotechnology Research InstituteShanghai Academy of Agricultural SciencesShanghaiChina
  2. 2.College of Bioscience and BiotechnologyYangzhou UniversityYangzhouChina

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