Abstract：
In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.
摘要：
鉴于几丁质酶（ChiA）在农业等各个领域的广泛潜在应用，环境保护,医学,和生物技术，开发能够产生具有增强活性的几丁质酶的高产菌株具有重要意义。本研究以苏云金芽孢杆菌胞外几丁质酶为研究对象,以地衣芽孢杆菌为表达宿主，实现活性增强的ChiA异源表达。最初，通过结构分析和分子动力学模拟，我们确定了改善几丁质酶酶性能的关键氨基酸，几丁质酶突变体D116N/E118N的比活性比天然酶高48%,伴随着热稳定性和pH稳定性的增强。随后，在地衣芽孢杆菌中筛选并修饰ChiA(D116N/E118N)的表达元件,导致细胞外ChiA活性达到89.31U/mL。进一步的努力涉及成功敲除细胞外蛋白酶基因aprE，bpra和epr,以及参与地衣芽孢杆菌的杆菌肽和地衣素等副产物合成的基因簇。这导致了重组菌株的发展，DW2△abelA，几丁质酶活性显着提高，达到145.56U/mL。为了进一步提高几丁质酶的活性，将几丁质酶表达框整合到DW2△abelA的基因组中，导致显著增加至180.26U/mL。进一步优化发酵条件和培养基组分,提高摇瓶酶活性,达到200.28U/mL，而放大发酵实验产生了338.79U/mL的令人印象深刻的酶活性。通过宿主基因改造,表达优化和发酵优化，成功构建了高产ChIA菌株，这将为ChiA的细胞外生产提供坚实的基础。
