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

壳聚糖是一种具有独特性质的生物聚合物，具有广泛的工业、生物医学和农业应用。壳聚糖是以罐头厂蟹壳下脚料中提纯的甲壳素为原料生产的。目前，甲壳素化学制取壳聚糖的过程中会产生大量的碱渣。如果甲壳素脱乙酰酶催化甲壳素转化为壳聚糖，预计不会产生任何废物。为了开发一种减少废物的酶法生产壳聚糖的工艺，我们计划从布氏藻类中分离出几丁质脱乙酰酶基因的克隆，该基因的细胞壁主要由几丁质和壳聚糖组成。根据毛霉几丁质脱乙酰酶基因中含有乙酰氨基的多糖脱乙酰酶基因中两个高度保守的序列，利用聚合酶链式反应从白毛霉基因组DNA中扩增出218bP-DNA片段，作为从我们构建的文库中分离克隆的探针。获得了1个全长为1,658bp的阳性克隆。该克隆编码的推导蛋白由451个氨基酸组成。蛋白质序列比较表明，该蛋白是一种与鲁氏支原体甲壳素脱乙酰酶有明显相似性的膜蛋白。这些结果表明，已成功地克隆了黑曲霉甲壳素脱乙酰酶基因。克隆的基因和编码的蛋白分别命名为PbCD和PbCDA。PbCD的核苷酸序列已在GenBank中注册(登录号：AB046690)。将该克隆与表达载体pET32Ek/LIC连接后，在纯化的大肠杆菌粗提物His标记的蛋白质组分中未检测到甲壳素脱乙酰酶活性，但表达了甲壳素脱乙酰酶。克隆所编码的蛋白对甲壳素脱乙酰酶活性表达的要求将是我们下一个需要阐明的项目。

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.