一种来源于喜热梭孢壳的耐热型β-甘露聚糖酶

Characterization of a hyperthermostable β-mannanase from Thermothelomyces thermophilus

摘要:

[目的] 本文旨在挖掘满足工业加工所需的新型耐热β-甘露聚糖酶。[方法] 从丝状真菌喜热梭孢壳中克隆甘露聚糖酶基因Mtman1，利用毕赤酵母表达系统进行异源表达，研究重组酶的酶学性质和热处理过程中蛋白构象变化情况。[结果] 序列分析显示，Mtman1基因编码409个氨基酸，包含一段20个氨基酸的信号肽序列和一个GH5家族结构域；经毕赤酵母表达的重组MtMAN1蛋白约为60 kDa，存在N-糖基化修饰，该酶的最适反应pH和温度分别为6.0和70℃，催化刺槐豆胶底物的K_m和V_max分别为4.28±0.73 mg/mL和203.9±14.61 μmol/（s·mg）；MtMAN1在60℃下十分稳定，在80℃、90℃和100℃下处理10 min后，分别能维持（68.23±7.47）%、（56.01±5.69）%和（14.91±2.92）%的残余活性，且二级结构和最大发射波长基本没有发生变化，提示其构象维持稳定；此外，该酶对较低浓度的Fe²⁺（<0.1 mmol/L）、Cu²⁺（<0.1 mmol/L）、Ca²⁺（<0.5 mmol/L）、Mg²⁺（<0.1 mmol/L）或Zn²⁺（<0.1 mmol/L）耐受能力较强。[结论] MtMAN1是一种耐热的β-甘露聚糖酶，在饲料制粒等需要高温处理的工业加工工艺中具有较好的应用前景。

Abstract:

[Objective] This study aims to obtain a novel thermostable mannanase to satisfy industrial application.[Methods] Sequence analysis revealed that the Mtman1 encodes for 409 amino acids (AAs), including a signal peptide and a glycoside hydrolase (GH) 5 catalytic domain. Recombinant MtMAN1 heterologously expressed in Pichia pastoris showed an optimal pH and temperature of 6.0 and 70℃, respectively. The K_m and V_maxvalues of MtMAN1 were 4.28±0.73 mg/mL and 203.9±14.61 μmol/s·mg against locust bean gum, respectively.[Results] The MtMAN1 was stable under 60℃ incubation for 1 h. After treatment at 80℃, 90℃ and 100℃ for 10 min, the enzyme maintained (68.23±7.47)%, (56.01±5.69)% and (14.91±2.92)% residual activities, respectively. Interestingly, its secondary structures and maximum wavelength remained as unchanged, suggesting relatively stable conformational structure. Additionally, the MtMAN1 was considerably resilient to Fe²⁺ (<0.1 mmol/L), Cu²⁺ (<0.1 mmol/L), Ca²⁺ (<0.5 mmol/L), Mg²⁺ (<0.1 mmol/L) or Zn²⁺ (<0.1 mmol/L).[Conclusion] MtMAN1 is a hyperthermostable β-mannanase and could be a candidate for handling industry tanks at high temperatures, such as feed pelleting.