Microbiology, School of Natural Sciences and National Centre for Biomedical Engineering Sciences, National University of Ireland, Galway, Ireland.
Appl Microbiol Biotechnol. 2010 Aug;87(5):1773-82. doi: 10.1007/s00253-010-2662-8. Epub 2010 May 23.
Beta-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable beta-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. beta-galactosidase crystal structure with bound beta-galactose. This led to targeted mutagenesis of an Asp(258)-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant beta-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K (i) of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K (m) (3.76 mM compared to 2.21 mM) and reduced V (max) (110.8 micromol min(-1) mg(-1) compared to 172.6 micromol min(-1) mg(-1)) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.
β-半乳糖苷酶是一种酶，作为消化补充剂用于治疗乳糖不耐受，这是一种在大多数世界地区普遍存在的遗传疾病。从黑曲霉 van Tiegh 中克隆出编码一种耐酸性β-半乳糖苷酶的基因，对其进行测序并在巴斯德毕赤酵母中表达。纯化的重组蛋白表现出与天然酶相似的动力学特性，因此在应用相关浓度下也受到其产物半乳糖的竞争性抑制。为了减轻这种产物抑制，根据与β-半乳糖结合的青霉属β-半乳糖苷酶晶体结构生成了该酶的结构模型。这导致在黑曲霉 van Tiegh 酶中靶向突变 Asp(258)-Ser-Tyr-Pro-Leu-Gly-Phe 氨基酸基序，并从所得文库中分离出对半乳糖抑制敏感性降低的突变β-半乳糖苷酶酶（半乳糖的 K (i) 为 6.46 mM，而野生型重组酶为 0.76 mM）。与野生型酶相比，突变酶还表现出增加的 K (m)（3.76 mM 比 2.21 mM）和降低的 V (max)（110.8 µm ol min(-1) mg(-1) 比 172.6 µm ol min(-1) mg(-1)），但其在模拟空腹胃条件下的稳定性显著降低。然而，该研究表明可以合理地对黑曲霉 van Tiegh 酶进行工程改造，以减轻产物抑制并创造出具有改善的、应用相关的动力学特性的突变体，用于治疗乳糖不耐受。
