Hu Mengkai, Liu Fei, Wang Zhi, Shao Minglong, Xu Meijuan, Yang Taowei, Zhang Rongzhen, Zhang Xian, Rao Zhiming
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Front Microbiol. 2022 Aug 10;13:979079. doi: 10.3389/fmicb.2022.979079. eCollection 2022.
Sucrose isomerase (SI), catalyzing sucrose to isomaltulose, has been widely used in isomaltulose production, but its poor thermostability is still resisted in sustainable batches production. Here, protein engineering and one-step immobilized cell strategy were simultaneously coupled to maintain steady state for long-term operational stabilities. First, rational design of SI (PdSI) for improving its thermostability by predicting and substituting the unstable amino acid residues was investigated using computational analysis. After screening mutagenesis library, two single mutants (PdSIV280L and PdSIS499F) displayed favorable characteristics on thermostability, and further study found that the double mutant PdSIV280L/S499F could stabilize PdSIWT better. Compared with PdSIWT, PdSIV280L/S499F displayed a 3.2°C-higher , and showed a ninefold prolonged half-life at 45°C. Subsequently, a one-step simplified immobilization method was developed for encapsulation of PdSIV280L/S499F in food-grade cells to further enhance the recyclability of isomaltulose production. Recombinant cells expressing combinatorial mutant (RCSI2) were successfully immobilized in 2.5% sodium alginate without prior permeabilization. The immobilized RCSI2 showed that the maximum yield of isomaltulose by batch conversion reached to 453.0 g/L isomaltulose with a productivity of 41.2 g/l/h from 500.0 g/L sucrose solution, and the conversion rate remained 83.2% after 26 repeated batches.
蔗糖异构酶（SI）可催化蔗糖转化为异麦芽酮糖醇，已广泛应用于异麦芽酮糖醇的生产，但在可持续批量生产中，其较差的热稳定性仍是一大阻碍。在此，蛋白质工程和一步固定化细胞策略同时联用，以维持长期操作稳定性的稳态。首先，利用计算分析研究了通过预测和替换不稳定氨基酸残基来合理设计SI（PdSI）以提高其热稳定性。筛选诱变文库后，两个单突变体（PdSIV280L和PdSIS499F）在热稳定性方面表现出良好特性，进一步研究发现双突变体PdSIV280L/S499F能更好地稳定PdSIWT。与PdSIWT相比，PdSIV280L/S499F的熔点高3.2℃，在45℃下的半衰期延长了9倍。随后，开发了一种一步简化固定化方法，将PdSIV280L/S499F包封在食品级细胞中，以进一步提高异麦芽酮糖醇生产的可回收性。表达组合突变体的重组细胞（RCSI2）无需预先通透处理就成功固定在2.5%的海藻酸钠中。固定化的RCSI2表明，从500.0 g/L蔗糖溶液中分批转化生产异麦芽酮糖醇的最大产量达到453.0 g/L，生产率为41.2 g/l/h，经过26次重复批次后转化率仍保持在83.2%。
