几丁质裂解性多糖单加氧酶的表达及应用研究

以具有几丁质降解能力菌株Chitiniphilus sp. LY72的裂解多糖单加氧酶（CsLPMO）为研究对象，在克隆CsLPMO全长基因的基础上对其进行生物信息学分析，利用3种表达质粒pET-22b、pET-28a和pCold，构建CsLPMO异源表达菌株EBL-22、EBL-28和EBL-Co。重组CsLPMO蛋白经Ni-NTA亲和层析柱纯化及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定，筛选最佳表达质粒，对其表达条件进行优化。结果表明，CsLPMO是一种裂解性多糖单加氧酶，隶属于AA10家族。3株重组菌中，纯化重组酶蛋白相对含量分别为5.9%、4.1%、5.4%，比酶活力分别为155.42 U/mg、80.26 U/mg、148.29 U/mg，因此，确定最佳质粒为pET-22b，菌株EBL-22表达条件为：OD600 nm值至0.4时，添加异丙基-β-D-硫代半乳糖苷（IPTG）至终浓度为0.4 mmol/L后，在25 ℃下诱导表达16 h。在优化条件下，其可溶性蛋白含量为928.32 mg/L，比酶活力为3.34 U/mL。CsLPMO的加入可使还原糖的产量提高1.67倍。

Expression and application of chitinolytic polysaccharide monooxygenase

Using lytic polysaccharide monooxygenase of Chitiniphilus sp. (CsLPMO) with chitin degradation ability as research object, bioinformatics analysis was carried out on the basis of cloning CsLPMO full-length gene. CsLPMO heterologous expression strains EBL-22, EBL-28 and EBL-Co were constructed using 3 kinds of expression plasmids pET-22b, pET-28a, and pCold. The recombinant CsLPMO protein was purified by Ni-NTA affinity chromatography column and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the optimal expression plasmids were screened, and their expression conditions were optimized. The results showed that CsLPMO was a lytic polysaccharide monooxygenase belonging to the AA10 family. In the 3 recombinant strains, the relative contents of purified ecombinase protein were 5.9%, 4.1%, and 5.4%, and the specific enzyme activities were 155.42 U/mg, 80.26 U/mg, and 148.29 U/mg, respectively. Therefore, the optimal plasmid was pET-22b. The expression conditions of strain EBL-22 were as follows: when OD600 nm value was 0.4, IPTG was added to the final concentration of 0.4 mmol/L and the expression was induced at 25 ℃ for 16 h. Under the optimized conditions, the soluble protein content was 928.32 mg/L, and the specific enzyme activity was 3.34 U/ml. The addition of CsLPMO increased the yield of reducing sugar by 1.67 times.