热紫链霉菌几丁质酶表达及低脱乙酰度壳寡糖制备

Expression of chitinase from Streptomyces thermoviolaceus and preparation of chitooligosaccharides with a low degree of deacetylation

壳寡糖具有抗炎、抗肿瘤及激活植物免疫等一系列生物活性.与传统的高脱乙酰度壳寡糖相比,低脱乙酰度壳寡糖可能具有更高生物活性.表达热紫链霉菌几丁质酶基因并使用表达产物水解制备低脱乙酰度壳寡糖;优化并全基因合成热紫链霉菌几丁质酶基因,利用毕赤酵母进行分泌表达,对产物酶的性质进行鉴定;利用表达的几丁质酶水解制备低脱乙酰度壳寡糖,使用超高效液相色谱-四极杆飞行时间质谱(Ultra-performance liquid chromatography quadrupoletime-of-f lightmassspectrometry,UPLC-QTOFMS)对其组分进行分离及鉴定,使用核磁共振(Nuclear magnetic resonance,NMR)确定其末端结构特征.结果显示,表达的几丁质酶蛋白浓度为0.20 mg/mL,最适pH为5.6,最适温度为60℃,酶活为0.98 U/mL,该酶在80℃及以下时较稳定,该酶水解制备的低脱乙酰度壳寡糖中包含至少35种聚合度2-17、不同脱乙酰度的壳寡糖组分,这些组分的还原末端主要由N-乙酰氨基葡萄糖组成,非还原末端同时包含氨基葡萄糖及N-乙酰氨基葡萄糖.本研究表明毕赤酵母分泌表达的热紫链霉菌几丁质酶具有较好的热稳定性,具有应用于低脱乙酰度壳寡糖规模制备的潜力.(图4表1参22)

Chitooligosaccharides have a range of biological activities, such as anti-inflammatory, anti-tumor, and activation of plant immunity. Compared with traditional high deacetylated chitooligosaccharides, low deacetylated chitooligosaccharides may have higher biological activity. In this study, the chitinase encoding gene of Streptomyces thermoviolaceus was expressed and chitooligosaccharides with a low degree of deacetylation were prepared by the expressed enzyme. The chitinase encoding gene of S. thermoviolaceus was optimized, synthesized, and secretorily expressed in Pichia pastoris. The properties of the produced enzyme were identified. Chitooligosaccharides with a low degree of deacetylation were prepared by the expressed enzyme and the composition and structural characteristics were analyzed by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance, respectively. The protein concentration of the expressed product reached 0.20 mg/mL. The optimum pH and temperature of the expressed chitinase was 5.6 and 60 ℃, respectively, and enzymatic activity reached 0.98 U/mL. The chitinase was continuously thermostable at 80 ℃. These hydrolysates contained at least 35 different kinds of chitooligosaccharides with a degree of polymerization of 2–17 and different degrees of deacetylation. The reducing end of oligosaccharide fractions were mainly composed of N-Acetylglucosamine, whereas both glucosamine and N-Acetylglucosamine were found in the non-reducing end. This study showed that the chitinase of S. thermoviolaceus secretorily expressed in P. pastoris had good thermal stability and has the potential to be used in the scale preparation of chitooligosaccharides with a low degree of deacetylation.