维氏气单胞菌来源几丁质酶的克隆表达及应用

Cloning, Expression and Application of Chitinase from Aeromonas veronii

丰年虾是一种小型甲壳类动物,孵化后的幼虫是水产动物的优质饵料,残留的卵壳则作为废弃物处理。然而丰年虾卵壳含有丰富的蛋白质和几丁质,具有极大的应用潜力。为了从丰年虾卵壳中制备几丁寡糖,选择了水解产物分布宽泛和含有几丁质结合域的维氏气单胞菌来源的几丁质酶ChiB565,将其在毕赤酵母Pichia pastoris中进行重组表达,对其酶学性质和高密度发酵条件进行研究后,建立了以中性蛋白酶和重组几丁质酶ChiB565为主协同处理丰年虾卵壳以制备几丁寡糖的绿色工艺。结果显示,重组菌发酵的最高酶活为13.2 U/mL,是摇瓶发酵酶活的3.88倍;当先以中性蛋白酶(30 mg/g(以底物质量计),4 h)处理丰年虾卵壳以脱除蛋白质,再用重组几丁质酶ChiB565(40 U/g(以底物质量计),5 h)来水解几丁质时,水解产物主要为几丁二糖到几丁四糖,伴随少量几丁五糖和几丁六糖,且水解产物对DPPH自由基和ABTS自由基清除率分别为59.94%和53.15%。上述研究表明以丰年虾卵壳为底物采用生物酶法制备几丁寡糖相较于传统化学法回收率更高,时间更短。该研究为几丁寡糖的工业化制备奠定了重要基础。

Artemia salina is a small crustacean. The hatched larvae are high-quality bait for aquatic animals, and the remaining eggshells are treated as waste. However, Artemia salina eggshells are rich in protein and chitin, which have great potential for application. To prepare chitin oligosaccharides from Artemia salina eggshells, chitinase ChiB565, which is derived from Aeromonas veronii, and has a wide distribution of hydrolysates and contains chitin binding domain, is selected and recombinantly expressed in Pichia pastoris. The enzymatic properties are studied and high-density fermentation is carried out. A green process for preparation of chitin oligosaccharides by synergistic treatment of Artemia salina eggshells with neutral protease and recombinant chitinase ChiB565 is established.The results show that the highest enzyme activity of recombinant bacteria fermentation is 13.2 U/mL,which is 3.88 times that of shake flask fermentation. When Artemia salina eggshells are treated with neutral protease(30 mg/g substrate and 4 h) first to remove protein, and then treatd with recombinant ChiB565(40 U/g substrate and 5 h) to hydrolyze chitin, products are mainly chitobiose to chitotetraose, with a small amount of chitopentaose and chitohexaose. And the scavenging ability of the hydrolysate to DPPH and ABTS are 59.94% and 53.15%, respectively. The studies above illustrat that the preparation of chitin oligosaccharides by biological enzymatic method using Artemia salina eggshells as substrate has a higher recovery rate and a shorter time than traditional chemical methods.This research has laid an important foundation for the industrial preparation of chitin oligosaccharides.