大肠杆菌表达几丁质酶对不同底物的酶学性质及其降解产物分析

Enzymatic properties of chitinase from recombinant Escherichia coli with different substrates and analysis of hydrolysates

采用重组大肠杆菌大量表达几丁质酶LICHI18A,经Ni-NTA柱纯化后,对不同底物的酶学性质进行了研究,以Lineweaver-Burk双倒数作图法测定米氏常数,采用薄层色谱(TLC)和高效液相色谱(HPLC)对降解产物进行分析。结果表明,该几丁质酶分子量为54 k Da,对水溶性壳聚糖的Km和Vmax分别为4.04 mg/m L和222.2μmol/(min·mg);对水溶性壳聚糖的反应最适温度和p H分别为60℃和7.0;对胶体几丁质的Km和Vmax分别为2.913 mg/m L和2.836μmol/(min·mg),反应最适温度和p H分别为40℃和5.0。Ni2+、Mg2+、Ca2+、Zn2+对胶体几丁质的降解有明显的促进作用,但对水溶性壳聚糖的降解没有促进作用,且Ni2+和Zn2+有一定的抑制作用。Tween-80对降解胶体几丁质起一定的促进作用,而不同浓度的SDS有抑制酶活作用,且浓度越高抑制作用越强。TLC和HPLC分析结果表明,该酶降解胶体几丁质的产物主要为几丁二糖和单糖,而水溶性壳聚糖的降解产物较为复杂,主要有单糖、几丁二糖和甲壳寡糖。

This study aimed to use chitinase which was over-expressed by Escherichia coli and purified by NiNTA column to study the differences of the enzymatic properties between different substrates,especially,colloidal chitin and water-soluble chitosan. The Lineweaver-Burk plot was employed to measure Michaelis-Menten Equations. The hydrolysates from different substrates were analyzed by Thin-Layer Chromatography( TLC) and High Performance Liquid Chromatography( HPLC). The results showed that the molecular weight of chitinase was 54 k Da. Km and Vmaxfor water-soluble chitosan were 4. 04 mg / m L and 222. 2 μmol /( min·mg),respectively. The optimum temperature and p H were 60 ℃ and 7. 0,respectively. Km and Vmaxfor the colloidal chitin were 2. 913 mg / m L and 2. 836 μmol /( min·mg),respectively. The optimum temperature and p H were 40 ℃ and 5. 0,respectively. Ni2 +,Mg2 +,Ca2 +and Zn2 +obviously enhanced the chitinase activity with colloidal chitin. But there was no enhancement effect with watersoluble chitosan. Ni2 +and Zn2 +showed inhibition effect. Tween-80 enhanced the chitinase activity with colloidal chitin,but SDS in different concentrations inhibited the activity. The analysis results from TLC and HPLC spectra showed that the main enzymatic hydrolysates from colloidal chitin were chitobiose and monosaccharides. The hydrolysates from water-soluble chitosan were more complicated,mainly including monosaccharides,chitobiose and chitooligosaccharides.