摘要
为实现生物质的高效降解,制备具有重要生理功能的阿魏酸,本实验从深绿木霉XS6发酵液中分离纯化深绿木霉阿魏酸酯酶(Trichoderma atroatroviride feruloyl esterase,TaFAE),并研究酶学特性。发酵液经(NH_4)_2SO_4沉淀、DEAE-Cellulose DE52阴离子交换层析和Sephadex G-200凝胶过滤层析后,得到电泳纯的TaFAE,比活力134.3 U/mg,回收率28.9%,纯化倍数31.52。经十二烷基硫酸钠-聚丙烯酰氨凝胶电泳测得酶分子质量约为66.4 kDa。TaFAE对阿魏酸甲酯亲和力最高,以其为底物,酶K_m值和V_(max)值分别为0.53 mmol/L和16.74 μmol/(min·mg)。以芥子酸甲酯为底物,酶促反应速率最大,达到32.21 μmol/(min·mg),是阿魏酸甲酯的2倍,对咖啡酸甲酯无活性,说明该酶具有严格的底物特异性。TaFAE最适pH值和温度分别为pH 4.0和40℃。在pH 2.0～9.0的范围内,30～40℃温度下都表现出良好的稳定性。金属离子Ca^(2+)和Mg^(2+)对TaFAE活性有显著激活作用,重金属离子Hg^(2+)和Pb^(2+)几乎完全抑制酶活性;β-巯基乙醇、二硫苏糖醇、十二烷基硫酸钠、Trition X-100增强酶活性,苯甲基磺酰氟有较强的抑制作用。木聚糖酶降解生物质的体系中加入TaFAE可显著增加阿魏酸和还原糖产量,表明TaFAE和木聚糖酶有良好协同作用。综上所述,TaFAE优良的耐酸性及其他酶学性质说明其在食品和饲料行业有较好的应用潜力。
In order to realize efficient biomass degradation in the preparation of ferulic acid, exerting important physiological functions in the body, feruloyl esterase(TaFAE) was isolated and purified from the fermentation broth of Trichoderma atroatroviride XS6 and its enzymatic characteristics were described in this experiment. The electrophoretically pure enzyme was obtained by consecutive ammonium sulfate precipitation, DEAE-cellulose DE52 anion exchange chromatography and Sephadex G-200 gel filtration chromatography. SDS-polyacrylamide gel electrophoresis(SDS-PAGE) of the enzyme revealed a single band of about 66.4 kDa. Its specific activity was 134.3 U/mg, with a recovery of 28.9%, and 31.52-fold purification. TaFAE had the highest affinity for methyl ferulate. When methyl ferulate was used as substrate, the Km and Vmax values of the enzyme were 0.53 mmol/L and 16.74 μmol/(min·mg), respectively. When methyl sultacide was used as substrate, the highest enzymatic reaction rate of 32.21 μmol/(min·mg) was obtained, which was twice as high as that of methyl ferulate. In addition, no activity was shown toward methyl caffeate, indicating that the enzyme has a stringent substrate specificity. The optimum pH and temperature of TaFAE were pH 4.0 and 40 ℃, respectively. In the pH range of 2.0–9.0 and at 30–40 ℃, TaFAE showed good stability. The metal ions Ca2+ and Mg2+ promoted TaFAE activity, whereas Hg^2+ and Pb^2+ almost completely inhibited the activity of the enzyme;β-mercaptoethanol, dithiothreitol(DTT), SDS, and Trition X-100 promoted the enzymatic activity, whereas PMSF had a strong inhibitory effect. Biomass conversion by xylanase produced significantly more ferulic acid and reducing sugar when TaFAE was added to the system, indicating that Ta FAE and xylanase have a good synergistic effect. In summary, TaFAE has good acid tolerance and enzymatic properties, which make it promising for applications in the feed and food industry.
作者
高兆建
许祥
王先凤
芦宁
王旭东
张铁柱
张抗震
焦巍
GAO Zhaojian;XU Xiang;WANG Xianfeng;LU Ning;WANG Xudong;ZHANG Tiezhu;ZHANG Kangzhen;JIAO Wei(College of Food (Biological) Engineering, Xuzhou Institute of Technology, Xuzhou 221018, China;Jiangsu Tianzhong Animal Husbandry Co. Ltd., Xuzhou 221018, China;Jiangsu Taihe Food Co. Ltd., Xuzhou 221018, China;Pizhou Golden Earth Fertilizer Co. Ltd., Xuzhou 221100, China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2019年第10期121-128,共8页
Food Science
基金
江苏省重点研发计划项目(BE2016316)
江苏省苏北科技计划项目(BC2013417
BN2015021)
徐州市科技计划项目(XF13C027)
