(天津科技大学 生物工程学院/工业发酵微生物教育部重点实验室, 天津 300457)
(College of Bioengineering/Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, China)
以Pichia pastoris GS115为宿主,对来源于特异腐质霉Humicolainsolens、棘孢曲霉Aspergillus aculeatus和黑曲霉Aspergillus niger的β-葡萄糖苷酶基因bglHi、bglAa和bglAn进行异源表达。以对硝基苯基-β-D-吡喃葡萄糖苷为底物,分析了3种酶的酶学性质,其中BglAn的酶活力最高,为90.83U/mg。3种酶的最适反应温度和最适pH值范围分别为55~65℃和5.0~6.0。在pH值为6.0、温度50℃、酶量2U的条件下,利用3种酶水解不同浓度的罗汉果苷Ⅴ。结果表明:不同来源的酶水解底物的特异性差别较大,其中BglHi和BglAa水解罗汉果苷Ⅴ生成罗汉果苷ⅢE的转化率较低,仅为5%~7%；而BglAn在底物质量浓度1mg/mL,反应20min时转化率为96.5%；提高底物质量浓度到5mg/mL,反应1h时转化率也可达97.9%,基本实现完全转化。通过酶法转化罗汉果苷Ⅴ,获得了较高纯度的产物罗汉果苷ⅢE,为后期开发不同结构、不同功能罗汉果苷类甜剂提供了新的途径。
The β-glucosidase gene bglHi, bglAa and bglAn, derived from Humicolainsolens, Aspergillus aculeatus and Aspergillus niger were heterologously expressed in Pichia pastoris GS115, respectively. The pNPG was selected as the substrate to investigate the properties of three enzymes.The optimum reaction temperature ranged from 55℃ to 65℃ while the pH value ranged from 5.0 to 6.0. The enzyme activity of BglAn was the highest, which was up to 90.83U/mg.Three enzymes with different concentrations were used to hydrolyze mogroside V under the conditions as follows:pH 6.0, temperature 50℃ and enzyme amount 2U. The results showed that there was significant difference among the substrate specificity of different β-glucosidases. The conversion rate of mogroside V to mogroside ⅢE of BglHi and BglAa was only 5%-7%. Meanwhile, the conversion of 1mg/mL substrates by BglAn was 96.5% when the enzymatic time was 20 min. When the concentration of substrate was increased to 5mg/mL, the conversion rate could reach 97.9% at 1h. This study could provide a novel strategy for the development of mogroside sweeteners with different structures and functions.