Microbial Biotechnology Laboratory, Department of Biotechnology, Panjab University, Chandigarh, India.
Indian J Microbiol. 2008 Sep;48(3):358-64. doi: 10.1007/s12088-008-0044-z. Epub 2009 Mar 25.
A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg(-1). It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K(m), k(cat) and catalytic efficiency (k(cat)/K(m)) values of recombinant chitinase were found to be 1.27 mg ml(-1), 0.69 s(-1) and 0.54 s(-1)M(-1) respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.
一种产壳聚糖酶的细菌——肠杆菌属 NRG4，以前在我们的实验室中被分离出来，已被报道具有广泛的应用，如抗真菌活性、真菌原生质体的产生以及从膨胀的壳聚糖中产生壳二糖和 N-乙酰-D-葡萄糖胺。在本文中，肠杆菌属壳聚糖酶的基因已被克隆并在大肠杆菌 BL21(DE3)中表达。壳聚糖酶基因的结构部分由 1686bp 组成。壳聚糖酶的推导氨基酸序列与粘质沙雷氏菌的壳聚糖酶具有高度的同源性（99.0%）。重组壳聚糖酶使用 His-Tag 亲和层析法纯化至近均一性。纯化的重组壳聚糖酶的比活性为 2041.6 U mg(-1)。它表现出与天然壳聚糖酶相似的性质，最适 pH 和温度分别为 5.5 和 45°C。使用膨胀的壳聚糖作为底物，重组壳聚糖酶的 K(m)、k(cat)和催化效率（k(cat)/K(m)）值分别为 1.27 mg ml(-1)、0.69 s(-1)和 0.54 s(-1)M(-1)。与天然壳聚糖酶一样，重组壳聚糖酶从膨胀的壳聚糖中产生具有药用价值的 N-乙酰-D-葡萄糖胺和壳二糖，并抑制许多真菌的生长。
