利用分子环化技术提高瘤胃微生物木聚糖酶热稳定性

Enhancing thermostability of xylanase from rumen microbiota by molecular cyclization

摘要:

在高温下保持催化活性是工业酶的重要性质。近年来，采用基因工程、蛋白质工程技术提高野生酶进行催化活性或耐热等性质取得了重要进展。文中利用新近建立起来的异肽键介导的SpyTag/SpyCatcher系统对瘤胃微生物来源的木聚糖酶XYN11-6进行分子环化，获得稳定的环化酶C-XYN11-6。在60 ℃、70 ℃和80 ℃下处理10 min，C-XYN11-6的残余活性为81.53%、73.98%和64.41%，分别是相同条件下线性蛋白L-XYN11-6残余活性的1.48、2.92、3.98倍。经60–90 ℃热处理10 min后，C-XYN11-6仍保持可溶状态，而L-XYN11-6几乎完全聚沉。内源荧光和8-苯胺-1-萘磺酸 (8-anilino-1-naphthalenesulfonic acid，ANS) 结合荧光光谱分析显示，较之L-XYN11-6，热处理环境中C-XYN11-6更能够维持其构象稳定。值得注意的是，分子环化提高了C-XYN11-6对0.1–50 mmol/L Ca2+或0.1 mmol/L Cu2+的耐受能力。综上所述，文中利用SpyTag/SpyCatcher系统获得热稳定性和离子稳定性提高的环化酶，为工业酶的分子改良及扩大其在工业领域的应用建立了良好基础。

Abstract:

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60–90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1–50 mmol/L Ca2+ or 0.1 mmol/L Cu2+ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.