Effect of Amino Acids of Substrate Binding Sites on -galactosidase Activity from Bacillus subtilis G1

Date Received: 07-06-2025

Date Published: 13-06-2025

##submissions.doi##: https://doi.org/10.31817/tckhnnvn.2013.11.6.

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Issue: Vol. 11 No. 6 (2013)

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Nhung, N., Thao, N., Hanh, V., Thi, Q., Hoa, V., & Duc, N. (2025). Effect of Amino Acids of Substrate Binding Sites on -galactosidase Activity from Bacillus subtilis G1 . Vietnam Journal of Agricultural Sciences, 11(6). https://doi.org/10.31817/tckhnnvn.2013.11.6.

Effect of Amino Acids of Substrate Binding Sites on -galactosidase Activity from Bacillus subtilis G1

Nguyen Thi Hong Nhung , Nguyen Thi Thao , Vu Van Hanh , Quyen Dinh Thi (*) , Vu Dinh Hoa , Nguyen Huu Duc

  • Tác giả liên hệ: [email protected]

    • Keywords

    Bacillus subtilis G1, -galactosidase, site-saturation mutagenesis

    Abstract


    b-Galactosidase (LacA) encoded by lacA gene from Bacillus subtilis G1 (Acession No EU585783) is a hydroltic enzyme that catalyzes the hydrolysis of lactose to produce lactose-free milk products. However, LacA has limited activity toward lactose. Therefore, we employed the method of site-saturation mutagenesis using PCR amplification with degenerate primers for generating mutants based on prediction of substrate binding sites to assess the effect of amino acids to the enzyme activity. Seven amino acid residues containing Arg120, Asn158, Trp331, Glu371, Lys372, Leu373 and His374 were identified as candidates for substrate binding sites using ExPASy program in Swiss-Prot. The screening results of the 7 libraries showed that these sites affect activity when ONPG was used as substrate. Most substitutions at these sites dramatically reduced the hydrolysis activity. Notably, 99.66% of colonies with Arg120 site showed  decrease in and loss of hydrolysis activity. The result suggested that Arg120 may play an important role in substrate recognition. Mutants at Arg120 substituted by Phe, Thr and Val showed complete loss of β-galactosidase activity.

    References

    Ly, H.D., Withers S.G. (1999). Mutagenesis of glycosidases. Annu Rev Biochem, 68: 487-522.

    Kang, S.K., Cho K.K., Ahn J.K., Bok J.D., Kang S.H., Woo J.H., Lee H.G., You S.K., Choi Y.J. (2005). Three forms of thermostable lactose-hydrolase from Thermus sp.IB-21: Cloning, expression, and enzyme characterization. J Biotechnol, 116: 337-346.

    Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino-acid sequence similarities. J Biochem, 280: 309–316.

    Dong, Y.N., Liu X.M., Chen H.Q., Xia Y., Zhang H.P., Zhang H., Chen W. (2011). Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis. J Dairy Sci, 94: 1176-1184.

    Placier, G., Watzlawick H., Rabiller C., Mattes R. (2009). Evolved β-Galactosidase from Geobacillus stearothermophilus with improved transgalactosylation yield for galacto-oligosaccharide production. Appl Environ Microbiol, 75: 6312-6321.

    Quyen, D.T., Nguyen T.T., Nguyen S.L.T., Vu V.H. (2011). Cloning, high-level expression and characterization of a b-galactosidase from Bacillus subtilis G1. Austr J Basic Appl Sci, 7: 193-199.

    Zheng, L., Baumann U., Reymond J.L. (2004). An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucl Acids Res, 32: 115.