内切甲壳素酶双重作用机制及酶法合成功能性低聚糖

Chitin-oligosaccharides (COS) exhibit diverse biological activities, including antimicrobial activity, outer-membrane permeabilizing activity, and prevention of pathogen adhesion to eukaryotic cells. Several of these activities are highly dependent on the molecular structure of oligosaccharides, however, studies on the relationship of oligosaccharide structure and function are scarce. The lack of studies on structure-function relationships of COS is partially explained by the lack of appropriate methodology to produce and to purify sufficient amounts of well-defined compounds. It is therefore the aim of this project to optimise the enzymatic production of COS with a defined degrees of polymerization and acetylation. Moreover, the diversity oligosaccharide structure for use in functional analyses will be extended by exploitation of the transglycosylation activity of chitinases. The biological function of COS will be evaluated with model systems to determine their effect on pathogen adhesion..This study aims to use Escherichia coli BL21 DE3 to heterologously express the chitinase LlChi18A and the chitin-binding protein LlCBP33A from Lactococcus lactis ssp. lactis with subsequent purification by Ni-NTA column to study the characteristics of the enzymology. N-Acetylated chitosans with different molecular weights and degrees of acetylation will be prepared by selective N-acetylation and hydrolysis by neutral protease. The hydrolysis mechanism of N-acetylated chitosans by chitinase will be investigated by variation of the parameters molecular weight, degree of acetylation, crystal structure and molecular size of the substrate on chitin hydrolysis. Enzymatic hydrolysis of chitin will be carried out by chitinase, or by synergistic activity of chitinase and the chitin binding protein to establish an optimal hydrolysis system. Transglycosylation activity of chitinase will be evaluated with hydrolysis product with different DP. COS as well as and, galactose lactose, or glucose will be considered as acceptor carbohydrolysates to produce hetero-oligosaccharides. The structure and composition of novel oligosaccharides obtained by chitinase-mediated transglycosylation will be analysed by liquid-chromatography - tandem mass spectrometry (HPLC/MS) and 13C-nuclear magnetic resonance. The anti-adhesive properties of the hydrolysates will be tested in an established model system using porcine strains of enterotoxigenic E. coli and porcine erythrocytes. This project will provide the theoretic basis of enzymatic hydrolysis of N-acetylated chitosan and conversion into functional oligosaccharides. It will be the first project to systematically exploit the transglycosylation activity of chitinases for the production of hetero-oligosaccharides with novel biological activities.

由于结构和活性之间的密切关系和低聚糖的功能性，人们越来越期望得到设想结构和链长的功能性低聚糖。本项目旨在利用内切甲壳素酶的水解和转糖基双重作用合成功能性低聚糖，拟在E.coli BL21 DE3中大量表达内切甲壳素酶LlChi18A和甲壳素结合蛋白LlCBP33A，并进行分离纯化及酶学性质研究；通过均相N-酰化反应制备不同乙酰度的N-乙酰化壳聚糖，研究其分子结构参数对甲壳素酶水解N-乙酰化壳聚糖的影响，揭示其催化水解机制及甲壳素结合蛋白的协同作用；再利用甲壳素酶的糖基转移作用，以不同聚合度的水解产物、半乳糖/乳糖等为底物合成功能性低聚糖，采用高效液相色谱-质谱联用和核磁共振碳谱分析产物结构，弄清糖基转移反应的规律；测试并比较甲壳类低聚糖对猪致肠病大肠杆菌的抗粘连性能；为水解N-乙酰化壳聚糖甚至甲壳类天然多糖生物合成功能性低聚糖提供科学理论依据。

几丁质是地球上仅次于植物纤维素的第二大生物资源，几丁质的水解产物 N-乙酰胺基葡萄糖及几丁寡糖可广泛应用在医药、化工、食品、环境等许多领域，需求量日益增加。本项目利用Lactococus lactis ssp. lactis IL1403菌株中的几丁质酶基因构建大肠杆菌工程菌，表达、分离纯化重组蛋白LlChi18A，并以N-乙酰化壳聚糖等底物研究其酶学性质及产物，还利用该酶降解废菌丝体中几丁质。利用甲壳素酶的水解作用和糖基转移作用，以水溶性N-乙酰化壳聚糖和乳糖为底物合成功能性低聚糖，表征其结构，测试并比较各低聚糖对猪致肠病大肠杆菌的抗粘连性能。研究结果表明：（1）重组E. coli BL21(DE3)/pEMT-ChiA的诱导条件优化，大量表达甲壳素酶并进行纯化，酶活性质研究表明：最适pH 3.7左右，最适温度37 ºC左右。1 mmol/L Na+、Li+、Mn2+、EDTA和5 mmol/L Ba2+对几丁质酶LlChi18A的活性有所增加。（2）几丁质酶LlChi18A对胶体几丁质的最适温度为40 ºC；最适pH为5，Km和Vmax分别为2.913mg•mL-1和2.836 μmol•min-1•mg-1。对水溶性N-乙酰化壳聚糖的最适温度为60 °C；最适pH为7.0，Km和Vmax值分别为4.04 mg•mL-1和222.2 μmol•min-1•mg-1，降解作用较强，降解速率较快，温度稳定性较好。Ni2+、Mg2+、Ca2+、Zn2+对胶体甲壳素的降解有明显的促进作用。但对水溶性壳聚糖的降解没有促进作用，且Ni2+和Zn2+有一定的抑制作用。（3）对不同底物的降解产物进行分析，该酶降解的主要产物为几丁二糖。对胶体几丁质的亲和力较好，降解产物主要为几丁二糖和单糖。对水溶性壳聚糖的降解效果较好，但降解产物较为复杂，主要有单糖、几丁二糖和甲壳寡糖。（4）采用酶法（几丁质酶和蜗牛酶的混合运用）将废菌丝体中的几丁质直接降解为几丁寡糖，其4天的产率为33.5%，为发酵工业废弃物的综合利用提供理论依据。(5) 甲壳素酶能水解水溶性壳聚糖得到甲壳二糖，转糖基到乳糖上主要得到GlcNAc-β-(1→4)-GlcNAc-β-(1 →[4 ])-Glu-β-(1→4)-Gal。对猪致肠病大肠杆菌ETEC K88，转糖基产物浓度为0.8g/L时，该功能性低聚糖具有较好活性。