Hydrolysis efficiency of β‐galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial β‐galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnβGal), Aspergillus oryzae (AoβGal), Bacillus circulans (BcβGal), Bifidobacterium bifidum (BbβGal), and Kluyveromyces lactis (KlβGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnβGal), Asn140Ala and Asn199Ala (AoβGal), Arg111Ala and Glu355Ala (BcβGal), Arg122Ala and Phe358Ala (BbβGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlβGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcβGal, BbβGal, and KlβGal. The double mutations in BcβGal and BbβGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlβGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of β‐galactosidases and rendering them promising for industrial applications.

中文翻译：

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具有工业重要性的微生物 β-半乳糖苷酶：点突变对乳糖水解反应影响的计算研究。

由于半乳糖的强烈抑制，β-半乳糖苷酶的水解效率受到影响，阻碍了乳糖的完全水解。减少这种抑制的一种替代方法是在酶的活性位点进行突变。本研究的目的是使用计算技术评估点突变对不同微生物 β-半乳糖苷酶活性位点的影响。的酶黑曲霉（AnβGal），米曲霉（AoβGal），环状芽孢杆菌（BcβGal），两歧双歧杆菌（BbβGal）和乳酸克鲁维酵母(KlβGal) 被使用。在距离半乳糖/乳糖分子最多 4.5 Å 的所有残基中进行突变，并计算结合能。突变体 Tyr96Ala (AnβGal)、Asn140Ala 和 Asn199Ala (AoβGal)、Arg111Ala 和 Glu355Ala (BcβGal)、Arg122Ala 和 Phe358Ala (BbβGal)、Tyr523Ala、Phe358Ala (BbβGal)、Tyr523Ala、Phe6l 和 5A12Gal 对 523Ala、Phe61 和 5K βGal 的结合效果最好对乳糖亲和力的影响较小。为了通过降低半乳糖亲和力来最大化酶反应，建议对 BcβGal、BbβGal 和 KlβGal 进行双重突变。BcβGal 和 BbβGal 的双突变导致半乳糖亲和力的最大降低，而 KlβGal 没有观察到令人满意的结果。使用计算工具，提出了降低半乳糖亲和力而不显着影响乳糖结合的突变体。