Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia.
Microb Cell Fact. 2011 Nov 4;10:94. doi: 10.1186/1475-2859-10-94.
Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.
A gene encoding a cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE) and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low temperatures.
By taking advantage of its high activity at low temperatures and its acidic pH optimum, this recombinant chitinase will be valuable in various biotechnological applications under low temperature and acidic pH conditions.
冷适应酶是嗜冷生物产生的蛋白质，在极低温度下表现出高催化效率。几丁质由不溶性同多糖β-（1，4）-连接的 N-乙酰葡萄糖胺组成，是自然界中第二丰富的生物聚合物。几丁质酶（EC 3.2.1.14）在自然界中几丁质的循环利用中发挥着重要作用。冷适应几丁质酶作用下的几丁质生物降解在工业应用中具有显著优势，例如在低温下处理富含几丁质的废物、在寒冷环境中生物防治植物病原菌以及生物防治冷藏食品的微生物腐败。
使用快速扩增 cDNA 末端（RACE）和 RT-PCR 技术从南极 Glaciozyma antarctica PI12 中分离出编码冷适应几丁质酶（CHI II）的基因。成功地在巴斯德毕赤酵母表达系统中表达了分离的基因。核苷酸序列分析表明，存在一个开放阅读框，长 1215bp，编码 404 个氨基酸的蛋白质。当在 25°C 的 BMMY 培养基中用 1%甲醇诱导时，重组几丁质酶被分泌到培养基中。SDS-PAGE 分析显示，纯化的重组几丁质酶有两条带，分别对应于非糖基化和糖基化蛋白，分子量约为 39 和 50 kDa。该酶具有酸性 pH 特性，最适 pH 值为 4.0，最适温度为 15°C。该酶在 pH 3.0-4.5 之间稳定，在 5-25°C 之间能够保持活性。K+、Mn2+和 Co2+离子的存在将酶活性提高了 20%。对不溶性底物的分析表明，纯化的重组几丁质酶对胶体几丁质具有很强的亲和力，对几丁聚糖几乎没有影响。与其他底物相比，CHI II 重组几丁质酶在低温下对胶体几丁质具有更高的 Vmax 和 Kcat 值。
利用其在低温下的高活性和酸性 pH 最佳值，这种重组几丁质酶将在低温和酸性 pH 条件下的各种生物技术应用中具有价值。
