Wenyingzhuangia属海洋细菌产紫菜多糖酶的克隆表达与性质及其在紫菜复合酶解中的应用

紫菜是一种重要的经济红藻,我国的紫菜产量高而产值较低。紫菜多糖是紫菜的主要组成部分,紫菜多糖及其降解产物被证实具有多种生理调节功能,是实现紫菜高值化开发及利用的重要途径。为挖掘紫菜多糖的工具酶并验证其应用的可行性,本研究对海洋细菌Wenyingzhuangia fucanilytica CZ1127T基因组中潜在的紫菜多糖酶编码序列por16B进行了异源表达、性质研究及应用探究。生化性质研究结果表明,重组蛋白Por16B_Wf的最适反应温度为40 °C,最适反应pH为7.0,且具有良好的贮存稳定性及pH稳定性。以高效液相色谱-质谱联用技术结合糖生物信息学的糖组学策略,明确了Por16B_Wf各个亚位点的作用方式。进一步将Por16B_Wf应用至紫菜多糖-蛋白复合酶解,与单纯蛋白酶酶解工艺相比,所得酶解液黏度低且紫菜转化率高。本研究为紫菜多糖的降解提供了生化性质及作用方式清晰的工具酶,并首次实现了紫菜的多糖-蛋白复合酶解,为紫菜的精深加工及高值化利用提供了新思路。

Expression and characterization of a porphyranase from marine bacterium Wenyingzhuangia fucanilytica and its application in compound enzymolysis of Pyropia

Pyropia is one of the most commercially important marine algae. Pyropia cultivation of China ranks first in the world, but the production value remains low. Porphyran is the major polysaccharide from Pyropia. Porphyran and its degradation products were confirmed to have various bioactivities, which is considered as an important way to realize the high-value application of Pyropia. In order to explore the glycoside hydrolase for degrading porphyran and verify the feasibility of its application, here, a porphyranase coding gene por16B was cloned from the marine bacterium Wenyingzhuangia fucanilytica and heterologously expressed in Escherichia coli. The biochemical properties and hydrolysis patterns of the recombinant protein Por16B_Wf were characterized. Por16B_Wf exhibited maximum activity at 40 °C and pH 7.0, and it possessed stable activity at different pH conditions. Por16B_Wf hydrolyzed porphyran by an endo-acting manner. The subsite specificity of Por16B_Wf was clarified by glycomics strategy. Furthermore, Por16B_Wf was applied in the compound enzymolysis of Pyropia. Compared to the classical enzymolysis technic of Pyropia using neutral protease, the compound enzymatic hydrolysate was characterized with low viscosity and high transformation rate of Pyropia. Por16B_Wf could be utilized as a promising tool for the degradation of porphyran. In this study, the compound enzymolysis of Pyropia was realized for the first time, which was beneficial to the full transformation of Pyropia, and provided new insights into the deep-processing and high-value utilization of Pyropia.