Alginate oligosaccharides are enzymolysis products of alginate with versatile bioactivities and their industrial preparation was limited by the insufficient activity and unsatisfying thermostability of alginate lyases. The structure-function information about PL18 alginate lyases was seldom reported since which few positive mutants of PL18 alginate lyases were generated. In present study, a mutant of PL18 alginate lyase E226 K was expressed intracellularly and taken as parent for further modification. Site I211 at the lid loop 1 and sites E276, Y292 and R294 at the predicted entrance were chosen as engineering targets based on the E226K-PM4 binding mode in prereaction-state MD simulation and 29 mutants were constructed, from those, the variant E226 K/I211 T/R294 V was screened as the best mutant (showing 4.78-fold increased catalytic efficiency and half-time t_1/2^45℃ increased up to 557 min from 89 min). MD simulations indicated that the affinity of E226 K/I211 T/R294 V towards alginate was improved due to the optimized energy distribution of active center, more flexible loops around catalytic cleft and larger substrate entrance. The more efficient proton transmitting endowed E226 K/I211 T/R294 V higher activity and the more complicated intraprotein interactions together with stronger backbone rigidity were responsible for the improved thermostability of E226 K/I211 T/R294 V than E226 K. The success in this study enriches the structure-function information of PL18 alginate lyases and provides hints for their further design.

海藻寡糖是海藻酸盐的酶解产物，具有多种生物活性，其工业制备受到海藻酸盐裂解酶活性不足和热稳定性不理想的限制。PL18 藻酸盐裂解酶的结构-功能信息很少被报道，因为很少产生 PL18 藻酸盐裂解酶的阳性突变体。在本研究中，PL18 海藻酸裂解酶 E226 K 的突变体在细胞内表达并作为亲本进行进一步修饰。根据反应前状态 MD 模拟中的 E226K-PM4 结合模式，选择盖子环 1 处的位点 I211 和预测入口处的位点 E276、Y292 和 R294 作为工程目标，并从中构建了 29 个突变体，其中，变体 E226 K /I211 T/R294 V 被筛选为最佳突变体（显示 4._1/2 ^45℃从 89 分钟增加到 557 分钟）。MD 模拟表明 E226 K/I211 T/R294 V 对藻酸盐的亲和力由于活性中心的优化能量分布、催化裂隙周围更灵活的环和更大的底物入口而提高。更有效的质子传输赋予 E226 K/I211 T/R294 V 更高的活性，更复杂的蛋白内相互作用以及更强的骨架刚性是 E226 K/I211 T/R294 V 比 E226 K 热稳定性提高的原因。该研究丰富了 PL18 藻酸盐裂解酶的结构-功能信息，并为其进一步设计提供了提示。