Identification and Immobilization of an Invertase With High Specific Activity and Sucrose Tolerance Ability of Gongronella sp. w5 for High Fructose Syrup Preparation
Identification and Immobilization of an Invertase With High Specific Activity and Sucrose Tolerance Ability of Gongronella sp. w5 for High Fructose Syrup Preparation
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DOI：
10.3389/fmicb.2020.00633
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发表时间：
2020-04
影响因子：
5.2
通讯作者：
Gang Zhou;C. Peng;Xiaosa Liu;Fei Chang;Yazhong Xiao;Juanjuan Liu;Zemin Fang
中科院分区：
文献类型：
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作者：
Gang Zhou;C. Peng;Xiaosa Liu;Fei Chang;Yazhong Xiao;Juanjuan Liu;Zemin Fang
转化酶催化蔗糖的水解为果糖和葡萄糖，可用作生产高果糖糖浆的替代品。在本研究中，我们报道了Gongronella sp.转化酶基因(GspInv)的异源表达。Komagataella Pastoris中的W5。甲醇诱导5d后，GspInv活性达到147.6±0.4U/mL。GspInv是一种蔗糖转化酶，对蔗糖的比活力为2,776.1±124.2 U/mg。GspInv对蔗糖(IC50=1.2M)、葡萄糖(IC50和GT；2 M)、果糖(IC50=1.5M)和多种金属离子具有很高的耐受性，使其成为生产高果糖糖浆的理想候选者。对糖类结合模块进行了序列优化，并将其与GspInv的N端融合。该融合蛋白在室温下吸附1h的固定化效率最高，每克纤维素吸附量可达8000U蛋白。纤维素固定化融合蛋白保留了GspInv的独特性质。以1M蔗糖为底物制备高果糖糖浆时，融合蛋白在填充床反应器中连续水解50h，蔗糖转化率保持在95%左右。融合蛋白还能完全降解甘蔗糖蜜中的蔗糖。我们的结果表明，GspInv是一种不寻常的转化酶，是制备高果糖糖浆的有前途的候选者。
Invertases catalyze the hydrolysis of sucrose into fructose and glucose and can be employed as an alternative in producing high fructose syrup. In this study, we reported the heterologous expression of an invertase gene (GspInv) of Gongronella sp. w5 in Komagataella pastoris. GspInv activity reached 147.6 ± 0.4 U/mL after 5 days of methanol induction. GspInv is invertase with a high specific activity of 2,776.1 ± 124.2 U/mg toward sucrose. GspInv showed high tolerance to sucrose (IC50 = 1.2 M), glucose (IC50 > 2 M), fructose (IC50 = 1.5 M), and a variety of metal ions that make it an ideal candidate for high fructose syrup production. A carbohydrate-binding module was sequence-optimized and fused to the N-terminus of GspInv. The fusion protein had the highest immobilization efficiency at room temperature within 1 h adsorption, with 1 g of cellulose absorption up to 8,000 U protein. The cellulose-immobilized fusion protein retained the unique properties of GspInv. When applied in high fructose syrup preparation by using 1 M sucrose as the substrate, the sucrose conversion efficiency of the fused protein remained at approximately 95% after 50 h of continuous hydrolysis on a packed bed reactor. The fused protein can also hydrolyze completely the sucrose in sugarcane molasses. Our results suggest that GspInv is an unusual invertase and a promising candidate for high fructose syrup preparation.