凝结芽孢杆菌耐热β­N­乙酰氨基葡萄糖苷酶在大肠杆菌的分泌表达及其在制备GlcNAc中的应用

Expression and characterization of β-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-β-D glucosamine production

摘要:

β-N-乙酰氨基葡萄糖苷酶体，可作用于几丁质或壳聚糖等天然底物，从末端水解产生N-乙酰-β-D氨基葡萄糖 (GlcNAc) 单体，其在医药和农业领域有较广泛的用途。文中克隆了耐热菌凝结芽孢杆菌Bacillus coagulans DMS1的β-N-乙酰氨基葡萄糖苷酶基因 (BcNagZ)，并成功在大肠杆菌Escherichia coli BL21(DE3) 进行了分泌表达，蛋白表达量达到0.76 mg/mL。纯化后的BcNagZ分子量为61.3 kDa，测得的比活力为5.918 U/mg；进一步对该酶进行表征，结果显示酶的最适反应pH为5.5，最适反应温度为75 ℃，在65 ℃处理30 min后还有85%的残余酶活力，表明该酶具有良好的热稳定性。该酶的米氏常数Km为0.23 mmol/L，Vmax为0.043 1 mmol/(L·min)。重组BcNagZ可以水解胶体几丁质得到微量的GlcNAc，可以将二糖水解为单糖；偶联已报道的外切几丁质酶AMcase，可以有效地将胶体几丁质水解为GlcNAc，得率达到86.93%。

Abstract:

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.