Enzymatic Production of High Molecular Weight Chitooligosaccharides Using Recombinant Chitosanase from Bacillus thuringiensis BMB171
Enzymatic Production of High Molecular Weight Chitooligosaccharides Using Recombinant Chitosanase from Bacillus thuringiensis BMB171
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DOI：
10.4014/mbl.1712.12012
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发表时间：
2018-03
期刊：
影响因子：
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通讯作者：
Lixin Kang;Si-jing Jiang;Lixin Ma
中科院分区：
文献类型：
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作者：
Lixin Kang;Si-jing Jiang;Lixin Ma
克隆了苏云金芽孢杆菌BMB 171的壳聚糖酶基因（btbchito），并在毕赤酵母中进行了异源表达。经纯化后，从1-1培养基中获得约300 mg重组壳聚糖酶，比活力为240 units/mg。通过薄层色谱法（TLC）和基质辅助激光解吸电离-飞行时间联合使用测定的结果MALDI-TOF质谱（MS）分析表明，壳聚糖经热裂解后得到的壳寡糖（COS）（N-脱乙酰基70%、80%和90%）通过rBTBCHITO水解的产物由低聚物组成，聚合度（DP）主要在三聚体至七聚体的范围内;还产生了高分子量壳五糖、壳六糖和壳七糖。还使用MS推断水解产物，因为C 0 S（n）是具有从一个到两个的各种乙酰基的复杂寡糖，因此未检测到非乙酰基C 0 S（GlcN）n和具有更多乙酰基（> 2）的C 0 S。利用该方法制备高分子量的壳聚糖，具有广泛的工业和生物学应用前景，壳聚糖酶的活性研究具有重要的应用价值。
The chitosanase gene (btbchito) of Bacillus thuringiensis BMB171 was cloned and heterologously expressed in the yeast Pichia pastoris. After purification, about 300 mg of recombinant chitosanase was obtained from the 1-1 culture medium with a specific activity of 240 units/mg. Results determined by the combined use of thin layer chromatography (TLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) showed that the chitooligosaccharides (COSs) obtained by chitosan (N-deacetylated by 70%, 80%, and 90%) hydrolysis by rBTBCHITO were comprised of oligomers, with degrees of polymerization (DP) mainly ranging from trimers to heptamers; high molecular weight chitopentaose, chitohexaose, and chitoheptaose were also produced. Hydrolysis products was also deduced using MS since the COSs (n) are complex oligosaccharides with various acetyl groups from one to two, so the non-acetyl COSs (GlcN)n and COSs with more acetyls (> 2) were not detected. The employment of this method in the production of high molecular weight COSs may be useful for various industrial and biological applications, and the activity of chitosanase has great significance in research and other applications.