Laboratory of Applied Microbiology, School of Pharmacy, School of Biological and Food Engineering, Changzhou University, Changzhou 213164, China.
Advanced Catalysis and Green Manufacturing Collaborative Innovation Center, Changzhou University, Changzhou 213164, China.
J Agric Food Chem. 2024 Feb 28;72(8):4358-4366. doi: 10.1021/acs.jafc.3c09313. Epub 2024 Feb 13.
The hydrolytic products of chitosanase from (SaCsn46A) were found to be aminoglucose and chitobiose, whereas those of chitosanase from (BsCsn46A) were chitobiose and chitotriose. Therefore, the sequence alignment between SaCsn46A and BsCsn46A was conducted, revealing that the structure of BsCsn46A possesses an extra loop region (194N-200T) at the substrate binding pocket. To clarify the impact of this loop on hydrolytic properties, three mutants, SC, TJN, and TJA, were constructed. Eventually, the experimental results indicated that SC changed the ratio of chitobiose to chitotriose hydrolyzed by chitosanase from 1:1 into 2:3, while TJA resulted in a ratio of 15:7. This experiment combined molecular research to unveil a crucial loop within the substrate binding pocket of chitosanase. It also provides an effective strategy for mutagenesis and a foundation for altering hydrolysate composition and further applications in engineering chitosanase.
（SaCsn46A）壳聚糖酶的水解产物被发现为氨基葡萄糖和壳二糖，而 （BsCsn46A）壳聚糖酶的水解产物则是壳二糖和壳三糖。因此，对 SaCsn46A 和 BsCsn46A 进行了序列比对，结果表明 BsCsn46A 的结构在底物结合口袋处具有一个额外的环区（194N-200T）。为了阐明该环对水解特性的影响，构建了三个突变体，SC、TJN 和 TJA。最终的实验结果表明，SC 将壳聚糖酶水解的壳二糖与壳三糖的比例从 1:1 改变为 2:3，而 TJA 则导致比例为 15:7。该实验结合分子研究揭示了壳聚糖酶底物结合口袋内的一个关键环。它还为突变提供了一种有效的策略，并为改变水解产物组成以及进一步在工程壳聚糖酶中的应用奠定了基础。
