摘要
依据密码子偏好性原则,对嗜热甘露聚糖酶manBK基因密码子进行优化,以提高嗜热甘露聚糖酶在毕赤酵母中表达量;并通过对该酶酶学性质的探究,探讨金属离子对酶稳定性影响及其在酶解魔芋甘露聚糖中的应用潜力。结果表明:嗜热甘露聚糖酶manBK基因优化后GC含量为40%~53%,平均值为47%,密码子适应指数(codon adaptationindex,CAI)为0.87,共有242个碱基进行优化,优化前后基因序列的一致性为76%。重组菌株在25℃条件下,诱导72 h发酵液中酶活力可达22 U/mL。甘露聚糖酶ManBK的最适反应温度为80℃,最适pH值为5.0,表观分子质量为45 kDa,动力学参数Km值为1.40 mg/mL,V(max)值为149.25μmol/(min·mg)。金属离子Fe^3+、Ca^2+、Mg^2+和Zn^2+对该酶活性有促进作用,分别使酶活力提高了32%、52%、23%和459%,而金属离子K^+、Li^+、Mn^2+和Cu^2+对ManBK酶有不同程度的抑制作用。进一步分析发现,Zn^2+能提高该酶的热稳定性,使70℃条件下的半衰期延长了116 min。当酶底比为1∶1时,反应温度70℃,pH 5.0反应进行1.5 h,魔芋聚糖即可达到较合适的酶解,魔芋聚糖降解物的抗氧化性能增强了5.6倍左右,ManBK酶在魔芋聚糖降解中具有极强的应用潜力。本研究为ManBK酶的制备及在魔芋深加工行业中的应用提供了一定的理论支撑。
In this study,we optimized the codon usage of the thermophilicβ-mannanase gene(manBK)and the fermentation conditions to increase its expression in Pichia pastoris.The enzymatic properties of ManBK were investigated and the effect of metal ions on the enzymatic stability was explored as well as its application potential in enzymatic hydrolysis of konjac mannan.Compared with the wild type manBK gene,242 base pairs were modified after the optimization,and the GC content decreased to 40%–53%,on average 47%.The codon adaptation index(CAI)was 0.87.The optimized synthetic sequence showed a 76%homology to the wild-type gene,which was successfully expressed in P.pastoris GS115 using the constitutive expression vector pPIC9K.The ManBK activity attained 22 U/mL after a 72-hour incubation at 25℃.The ManBK had a molecular mass of approximately 45 kDa.The optimum temperature and pH value for the enzyme activity were 80℃and pH 5.0,respectively.The kinetic parameters Km and Vmax of ManBK were found to be 1.40 mg/mL and 149.25μmol/(min·mg),respectively.The enzyme was strongly activated by Fe^3+(32%),Ca2+(52%),Cu^2+(23%)and Zn^2+(459%)but inhibited by K^+,Li^+,Mn^2+and Cu^2+.In addition,Zn^2+enhanced the thermal stability of the enzyme,which prolonged the half-life time by 116 min at 70℃.The optimal conditions for enzymatic hydrolysis of konjac flour were determined as follows:reaction time 1.5 h,reaction temperature 70℃,pH 5.0,and E/S ratio 1:1.The antioxidant activity of the konjac degradation products was 6.6 times as high as that of konjac flour.In conclusion,The ManBK has a great potential application in the preparation of konjac oligosaccharide.
作者
卢海强
谷新晰
袁巧敏
谈苏慧
田洪涛
LU Haiqiang;GU Xinxi;YUAN Qiaomin;TAN Suhui;TIAN Hongtao(College of Food Science and Technology,Hebei Agricultural University,Baoding 071000,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2020年第18期113-119,共7页
Food Science
基金
国家自然科学基金青年科学基金项目(31501417)
河北省食品科学与工程学科“双一流”建设资金项目(2016SPGCA18)。
关键词
甘露聚糖酶
魔芋聚糖降解物
密码子优化
热稳定性
抗氧化性
mannanase
konjac degradation products
codon optimization
thermal stability
antioxidant activity
