Cloning of chitin deacetylase gene from Phycomyces blakesleeanus and its industrial application

壳聚糖是一种具有独特性能的生物聚合物，具有广泛的工业、生物医学和农业应用前景。壳聚糖是从制罐厂的蟹壳废料中提纯出来的几丁质为原料制备的。目前甲壳素制壳聚糖的化学过程中产生了大量的碱性废物。如果几丁质通过几丁质脱乙酰酶转化为壳聚糖，预计不会产生废物。为了开发一种减少壳聚糖浪费的酶促生产工艺，我们计划从主要由壳聚糖和壳聚糖组成的黑藻细胞壁中分离出一个几丁质脱乙酰酶基因克隆。用PCR引物从黑毛霉基因组DNA中扩增出218bp-DNA片段，该片段与rouxii毛霉几丁质脱乙酰酶基因中乙酰胺基的多糖脱乙酰酶高度保守的两个序列相连接，作为从□ZAP载体上构建的cDNA文库中分离克隆的探针。分离得到1 658bp的阳性cDNA克隆。该克隆编码的推断蛋白由451个氨基酸组成。蛋白质序列比较表明，该蛋白为一种膜蛋白，与rouxii的几丁质去乙酰化酶有显著的相似性。结果表明，几丁质脱乙酰酶基因已成功克隆。克隆的基因被命名为PbCD，该基因编码的蛋白被命名为pbda。PbCD的核苷酸序列已注册到GenBank（登录号：AB046690）。将克隆与表达载体pET32 Ek/LIC连接后，大肠杆菌转化粗提物中纯化的带有his标记的蛋白片段未检测到几丁质去乙酰化酶活性，但表达得到了鉴定。克隆编码的蛋白对几丁质脱乙酰酶活性表达的要求将是我们下一步要阐明的项目。

Chitosan is a biopolymer with unique properties favorable for a broad variety of industrial, biomedical, and agricultural application. The chitosan is produced from chitin which is purified from crab shell waste of can factories. Chemical procedure of chitin to chitosan leaves a lot of alkaline waste at present. If chitin is enzymatically converted to chitosan by chitin deacetylase, no waste is expected to be produced. To develop an enzymatic process for chitosan production that reduces the waste, we have planned to isolate a clone of chitin deacetylase gene from Phycomyces blakesleeanus in which cell wall is composed of mainly chitin and chitosan. The 218bp-DNA fragment amplified from genomic DNA of P. blakesleeanus using PCR primers to two highly conserved sequences among deacetylase for polysaccharide with acetoamido groups within chitin deacetylase gene of Mucor rouxii was the probe for isolation the clone from the cDNA library which we made in the □ZAP vector. A positive cDNA clone which is composed of 1, 658bp was isolated. The deduced protein encoded by this clone is composed of 451 amino acids. The protein sequence comparison revealed that the protein is a membrane protein significantly similar to chitin deacetylase of M. rouxii. These results show that the chitin deacetylase gene has been successfully cloned from P. blakesleeanus. The cloned gene and the putative protein encoded by the gene were named PbCD and PbCDA respectively. The nucleotide sequence of PbCD was registered to GenBank (accession #AB046690). After ligation of the clone to the expression vector pET32 Ek/LIC, chitin deacetylase activity was not detected in the purified His-tagged protein fraction of the crude extract from a E. coli transformant though the expression was identified. Requirements of the protein encoded by the clone for the expression of chitin deacetylase activity will be our next project to be elucidated.