Abstract
Sucrose isomerase (SI), catalysis sucrose to isomaltulose, has been wildly used in industrial production of isomaltulose. Here, rational design of Pantoea dispersa SI for improving its thermostability by predicting and substituting the unstable amino acid residues was studied using the computational-aided predictor FoldX. Through the mutation pool, two mutants of SI (V280L, S499F) displayed favorable characteristics on thermostability. The double mutant V280L/S499F were further constructed and showed prolonged half-life at 45 ℃, about 9-fold compared to the wild-type. Accordingly, the melting temperature of mutant V280L/S499F was improved to 54.2 ℃. To determine the recyclable ability of mutant V280L/S499F to bioconversion of isomaltulose, recombinant Corynebacterium glutamicum/ pXMJ19/pdsi V280L/S499F was constructed and repeated batch conversion was performed in a 5 L bioreactor. The results shown that the maximum yield of isomaltulose by batch conversion reached to 451 g/L with a productivity of 45.1 g/L/h, and the conversion rate remained 83.2 ± 2.1% even after 15 repeated batches of biocatalysis. Structure-based molecule molding revealed that the interiors of mutant V280L/S499F was more tightly packed in ɑ-9 fold and a new hydrophobic network was formed in ɑ-17, which combined contributed to improving its thermostability. This work provides new reference for the sustainable production of isomaltulose.
Introduction
Isomaltulose, a sucrose isomer, is a reducing disaccharide that exists in natural molasses in a small amount and has a sweetness of 45% sucrose but is nontoxic and non-cariogenic.[1]Therefore, isomaltulose is an ideal sucrose substitute and is the healthy sugar certified by United States Food Drug Administration, its addition and consumption are not restricted.[2] In addition, isomaltulose has many beneficial healthcare functions and physiological properties, including inhibiting elevated blood sugar levels,[3] inhibiting fat accumulation,[4] improving anti-fatigue ability,[5] and maintaining the intestinal microecological balance.[6] However, the process of chemically synthesizing isomaltose produces by-products and chemical waste, which increases the cost of product separation and wastewater treatment.[7] Therefore, the preparation of isomaltulose by biotransformation technology has been widely investigated in recent years.
Sucrose isomerase (SI, EC 5.4.99.11), also be known as isomaltulose synthase, is responsible for conversing sucrose into isomaltulose or trehalulose along with glucose and fructose.[8]Current investigations of SI are mainly in the mining of novel genes and property characterizations of SIs. Those currently-reported SIs showed limited thermostability during the biocatalysis process, such as the SI of Klebsiella sp. LX3, which has a 1.8 min half-life at 50 °C,[9] SI of Klebsiella pneumonia lost its 40% relative activity after incubating at 50 °C for 20 min,[10] and SI of Erwinia sp . was completely inactive after incubating at 30°C for 24 h.[11, 12] Although most studies have used enzyme immobilization or cell surface display technology to improve the robustness of SI, their industrial applications are still subject to unsatisfactory thermostability, and the conversion rate often decline rapidly after several continuous rounds of biocatalysis. Therefore, modification at the molecular level to improve the thermostability of SI should be further investigated.
Protein engineering has proven to be an effective approach to enhance thermostability of enzymes. Protein engineering is subdivided into directed evolution (irrational design), semi-rational design and rational design.[13] Although irrational design and semi-rational design were powerful in enzyme modification at elevated temperature,[14, 15] they are time-consuming and laborious. In contrast, rational design based on computer-aided techniques has greatly accelerated the speed and success rate of scientific research.[16] Fold X, one of the most reliable computational design predictors, has been developed to predict beneficial substitutions related to thermal stability by performing a rapid evaluation of the Gibbs free energy difference (ΔΔG)[17, 18]. Recently, FoldX has been used to improve the thermostability of many enzymes by rational design. Luo et al . obtained a best variant PoOPHM9 with a thermostability (T5015) of 67.6℃ by hierarchical iteration mutagenesis using FoldX.[19] Bi et al . engineered thermophilic pullulanase by rational design using FoldX predictor, theTm of mutant G692M increased by 3.8 °C,and half-life is 2.1-fold longer than the wild-type at 70 °C.[20] Wang et al . constructed a quadruple mutation (S142A/D217V/Q239F/S250Y) variant based on FoldX algorithm, the half-life of combination mutant increased 41.7-fold at 60 °C.[21] Thus, in-silico energy calculations (FoldX) may provide a clear guide for the molecular engineering of SI.
Compare with free-enzyme catalysis, while whole-cell biotransformation provides unique advantages, such as lower-cost preparation, easier separation of products and simpler recycle course. However, endotoxin or toxic cell wall pyrogens of non-food-grade host would be an obstacle to green synthesis of isomaltulose. To solve the potential safety hazards, some researchers have introduced SIase genes into non-pathogenic hosts, including Lactococcus lactisMG1363,[22] B. subtilisWB800,[23]Saccharomyces cerevisiae, [24] Yarrowia lipolyticaS47.[25] However, recombinant L. lactisMG1363 exhibited low expression level of sucrose isomerase (100 μg/mL), and Saccharomyces cerevisiae and Yarrowia lipolytica grew slowly (48-96h). Corynbacterinum glutamicum ATCC13032 (C. glutamium 13032) is listed as a “generally recognized as safe” microorganism and has been successfully used as a host for producing food compounds efficiently, like amino acids, vitamins and organic acids and rare sugars.[26] Furthermore, C. glutamicum has many advantages and is superior to other food-grade strains, such as non-pathogenic, non-codon bias and short fermentation period. Thus, isomaltulose production by whole-cell biotransformation using an ideal food-grade host was expected to be more suitable in the field of food and fermentation research.
Herein, in order to obtain robust SI from a small mutation library via rational design, computational design software (FoldX5) coupled with conservation analysis and functional region assessment were employed to predict potential stabilizing point mutations. Then, the best variant was intracellular overexpressed in the food-grade strain C. glutamium 13032, and the recombinant cells was further used as whole-cell biocatalyst for the recyclable synthesis of isomaltulose under the optimized conditions. Finally, Differential Scanning Fluorimetry (DSF) was used to evaluate the changes in the thermostability, and molecular dynamic simulation (MD) was used to elucidate the mechanism for the improved stability. Taken together, this study provides a new strategy for enhancing the stability of sucrose isomerase to improve its performance in industrial applications.
Materials and methods