Zhang Keke, Yang Yan, Wang Weidong, Liu Weizhi, Lyu Qianqian
MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China.
Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China.
J Agric Food Chem. 2021 Jun 16. doi: 10.1021/acs.jafc.1c02473.
Recently, we reported alginate lyase AlyF that predominantly produced trisaccharides (the trisaccharide content is 87.0%), and the determination of its substrate-binding mode facilitated its protein engineering for new product distribution. To clarify the relationship between the substrate-binding pocket and end-product distribution, the open binding pocket change was initially designed. The resulting F128T_W172R mutant of AlyF exhibited different intermediate-product distributions but still similar end-product distributions. However, these observations suggested that cleavage pattern changes for intermediate products might contribute to an altered end-product distribution. Structural analysis indicated that the sugar-binding affinity at subsite -2 should be redesigned to achieve this goal. Thus, residue Arg266, which is involved in sugar binding at subsite -2, was selected for site-saturation mutagenesis in the F128T_W172R mutant. The dominant end products of the F128T_W172R_R226H mutant were altered to disaccharides and trisaccharides (the disaccharide content increased to 40.5%).
最近，我们报道了主要产生三糖（三糖含量为87.0%）的海藻酸盐裂解酶AlyF，对其底物结合模式的确定有助于对其进行蛋白质工程改造以实现新的产物分布。为了阐明底物结合口袋与终产物分布之间的关系，最初设计了开放结合口袋的变化。所得的AlyF的F128T_W172R突变体表现出不同的中间产物分布，但终产物分布仍相似。然而，这些观察结果表明，中间产物的切割模式变化可能导致终产物分布改变。结构分析表明，应重新设计-2亚位点的糖结合亲和力以实现这一目标。因此，选择参与-2亚位点糖结合的残基Arg266在F128T_W172R突变体中进行位点饱和诱变。F128T_W172R_R226H突变体的主要终产物变为二糖和三糖（二糖含量增加到40.5%）。
