Tews I, Vincentelli R, Vorgias C E
European Molecular Biology Laboratory, c/o DESY, Hamburg, Germany.
Gene. 1996 Apr 17;170(1):63-7. doi: 10.1016/0378-1119(95)00848-9.
The chitobiase (Chb) encoding gene (chb) from Serratia marcescens (Sm) has been cloned, sequenced and expressed in Escherichia coli (Ec). Sequencing has revealed an open reading frame encodinga protein of 885 amino acids (aa). Ec cells harbouring plasmids containing chb can produce enzymatically active Sm Chb protein which is secreted into the periplasm. An efficient purification scheme using cation-exchange chromatographyis presented. This yields about 3 mg of > 95% pure Sm Chb per litre of Ec culture. The deduced aa sequence is 27-aa longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the cell membrane. Comparison with the other members of the family 20 of glycosyl hydrolases revealed that Chb has a conserved central region which aligns with almost all members of this family. According to the crystal structure of Sm Chb, this region comprises the catalytic domain of Chb which has an alpha/beta barrel fold.
粘质沙雷氏菌（Sm）的壳二糖酶（Chb）编码基因（chb）已被克隆、测序并在大肠杆菌（Ec）中表达。测序揭示了一个编码885个氨基酸（aa）蛋白质的开放阅读框。携带含有chb质粒的Ec细胞能够产生具有酶活性的Sm Chb蛋白，该蛋白分泌到周质中。本文介绍了一种使用阳离子交换色谱的高效纯化方案。每升Ec培养物可产生约3毫克纯度大于95%的Sm Chb。推导的氨基酸序列在N端比纯化蛋白测序确定的序列长27个氨基酸，这表明在酶跨细胞膜转运过程中一个前导序列被去除。与糖基水解酶家族20的其他成员比较表明，Chb有一个保守的中央区域，该区域与该家族几乎所有成员对齐。根据Sm Chb的晶体结构，该区域包含具有α/β桶状折叠的Chb催化结构域。
