Gal S W, Choi J Y, Kim C Y, Cheong Y H, Choi Y J, Bahk J D, Lee S Y, Cho M J
Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, South Korea.
FEMS Microbiol Lett. 1997 Jun 15;151(2):197-204. doi: 10.1111/j.1574-6968.1997.tb12570.x.
A DNA fragment (pCHI5422) containing two genes encoding a 54-kD and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)2 dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 microM (min)-1 mg-1 and 17 microM (min)-1 mg-1 on the natural swollen chitin, respectively.
从粘质沙雷氏菌KCTC2172的黏粒DNA文库中分离出一个含有两个基因的DNA片段（pCHI5422），这两个基因分别编码一种54-kD和一种22-kDa的几丁质酶。测定了由4581 bp组成的pCHI5422的完整核苷酸序列。22-kDa几丁质酶的核苷酸序列由681 bp的开放阅读框组成，编码227个氨基酸，位于54-kDa几丁质酶序列翻译终止密码子下游1422 bp处。54-kDa几丁质酶基因由1497 bp组成，位于一个单一的开放阅读框中，编码499个氨基酸。编码54-kDa和22-kDa几丁质酶的基因分别在大肠杆菌中进行亚克隆，并使用几丁质亲和色谱法从培养液中表达和纯化出各自的几丁质酶。当以壳六糖作为底物时，54-kDa和22-kDa几丁质酶的酶促反应的主要产物都是一种（GlcNAc）2二聚体，还有少量单体。54-kDa和22-kDa几丁质酶对天然膨胀几丁质的比活性分别为300 μmol·（min）-1·mg-1和17 μmol·（min）-1·mg-1。
