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Protein engineering of a cold-active β-galactosidase from Arthrobacter sp. SB to increase lactose hydrolysis reveals new sites affecting low temperature activity

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Abstract

We examined variants of an especially cold-active β-galactosidase (BgaS) to better understand features affecting enzyme activity at temperature extremes. We targeted locations corresponding to a region in the LacZ enzyme previously shown to increase activity and decrease thermostability. Changes in this region of BgaS consistently caused the elimination or reduction of activity. A gene (bgaS3) encoding a loss of function variant was subjected to random mutagenesis to restore activity and discover potential interactions important in cold activity. Gene sequences from the resulting library indicated that only two amino acid alterations, E229D and V405A, were required to restore activity. Genes with combinations of these mutations were constructed and their enzymes purified. Enzymes with the E229D/V405A/G803D alterations (BgaS6), or E229D/V405A (BgaS7) had similar thermal optima and thermostabilities as BgaS. BgaS7, however, showed a 2.5-fold increase in catalytic activity at 15°C and hydrolyzed 80% of lactose in skim milk in less than half the time of BgaS at 2.5°C. Computer-generated models predicted that the substitutions at positions 229 and 405 yielded fewer contacts at the enzyme’s activating interface. Results from regional saturation mutagenesis supported this hypothesis and suggested that not easily predicted, subtle, cooperative intramolecular interactions contributed to thermal adaptation.

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Acknowledgments

We thank members of our research group for helpful discussions and suggestions. We would also like to thank Kevin Gutshall for his help with the directed mutagenesis, and Al Phillips for discussions concerning the kinetics experiments and the manuscript. This work was supported by Department of Energy grant DE-FG02-93ER20117. James Coker was partially supported by NSF Research Training Grant DBI-9602232.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA

    James A. Coker & Jean E. Brenchley

  2. Center of Marine Biotechnology, 701 East Pratt Street, Baltimore, MD, 21202, USA

    James A. Coker

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  1. James A. Coker
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  2. Jean E. Brenchley
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Correspondence to James A. Coker.

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Communicated by J. Wiegel

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Coker, J.A., Brenchley, J.E. Protein engineering of a cold-active β-galactosidase from Arthrobacter sp. SB to increase lactose hydrolysis reveals new sites affecting low temperature activity. Extremophiles 10, 515–524 (2006). https://doi.org/10.1007/s00792-006-0526-z

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