Institute for Biology VII, Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
Advanced Environmental Research Laboratories, Department of Biology-Chemistry, West University of Timisoara, Oituz 4, 300086 Timisoara, Romania.
Int J Mol Sci. 2021 Mar 16;22(6):3041. doi: 10.3390/ijms22063041.
Chitinases catalyze the degradation of chitin, a polymer of -acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the DSM8785 chitinase A () gene in BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-,',″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term expression in by limiting the reaction time. We identified 12 mutants containing 2-8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.
几丁质酶催化几丁质的降解，几丁质是甲壳质、昆虫外骨骼和真菌细胞壁中 -乙酰氨基葡萄糖的聚合物。人们对开发改良的几丁质酶非常感兴趣，以解决食品加工工业中几丁质废物带来的环境负担，以及部分脱乙酰化几丁质（壳聚糖）及其产品（壳寡糖）的潜在医学、农业和工业用途。几丁质的解聚可以通过化学和物理处理来实现，但酶法过程会更环保、更可持续。然而，几丁质酶是作用缓慢的酶，限制了它们在生物技术中的应用，尽管通过分子进化方法来增强特定应用所需的特性可以克服这一限制。本研究的两个主要目标是开发一种用于几丁质酶活性的高通量筛选系统（该系统可推广到其他水解酶），并利用这种新方法来选择改良的几丁质酶变体。为此，我们在 BL21（DE3）细胞中克隆和表达了 DSM8785 几丁质酶 A（）基因，并通过易错 PCR 生成了突变文库。然后，我们开发了一种基于荧光激活细胞分选（FACS）的筛选方法，使用模型底物 4-甲基伞形酮基-β-D-，'，''-三乙酰基壳三糖苷来鉴定改良酶。我们通过在水相中环糊精来防止由酶反应的荧光产物 4-甲基伞形酮的疏水性引起的乳液隔室之间的串扰。我们还通过限制反应时间来解决在 中进行长期表达的毒性问题。我们鉴定了 12 个突变体，每个基因含有 2-8 个突变，导致酶活性比野生型 ChiA 高 2 倍。
