Alsina Cristina, Faijes Magda, Planas Antoni
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull Via Augusta 390, 08017, Barcelona, Spain.
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull Via Augusta 390, 08017, Barcelona, Spain.
Carbohydr Res. 2019 May 15;478:1-9. doi: 10.1016/j.carres.2019.04.001. Epub 2019 Apr 12.
Chitooligosaccharides (COS), the depolymerization products of chitin, have many potential applications in agriculture and medicine since they induce immunostimulating effects and disease protective responses. Most of their biological activities require degrees of polymerization (DP) larger than the tetrasaccharide, but structurally well-defined COS with DP larger than six are difficult to produce due to their high insolubility and complex isolation from chitin hydrolysates. Enzymatic synthesis by exploiting the transglycosylation activity of chitinases offers a potential strategy for the assembly of oligomers in the range of bioactive DPs. We here explore the glycosynthase-like activity of six GH18 chitinases from bacterial and archaeal origin by mutating the catalytic assisting residue in the substrate-assisted mechanism of this enzyme family. The alanine mutants at the assisting residue have a significant, but not essential, effect on the hydrolase activity. We studied the ability of the alanine mutants at the assisting residue to catalyze the polymerization of an oxazoline derivative as donor substrate, selecting the oxazoline of pentaacetylchitopentaose (DP5ox) with the aim of obtaining larger oligomers/polymers that, being insoluble, might be resistant to further reactions by the hydrolytically compromised mutant enzymes. For all the enzymes, insoluble polymeric material was obtained, with DP10 as major component, but other COS with different DPs were also obtained, limiting the practical application to produce oligomers/polymers with a defined DP. The balance between the residual hydrolase activity of the mutant enzymes and the solubility/precipitation kinetics still lead to hydrolysis and/or transglycosylation reactions on the newly formed products. From the selected enzymes, the Thermococcus kodakaraensis ChiA D1022A mutant gave the best results, with the formation of insoluble polymers in 45% yield (w/w) and containing about 55% of the target DP10 product.
壳寡糖（COS）是几丁质的解聚产物，由于其具有免疫刺激作用和疾病保护反应，在农业和医学领域有许多潜在应用。它们的大多数生物活性需要聚合度（DP）大于四糖，但由于其高不溶性以及从几丁质水解物中分离复杂，难以生产出结构明确且DP大于六的COS。利用几丁质酶的转糖基化活性进行酶促合成，为组装具有生物活性DP范围的寡聚物提供了一种潜在策略。我们在此通过突变该酶家族底物辅助机制中的催化辅助残基，探索了六种来自细菌和古菌的GH18几丁质酶的类糖基合酶活性。辅助残基处的丙氨酸突变体对水解酶活性有显著但非必需的影响。我们研究了辅助残基处的丙氨酸突变体催化恶唑啉衍生物作为供体底物聚合的能力，选择了五乙酰壳五糖恶唑啉（DP5ox），目的是获得更大的寡聚物/聚合物，由于其不溶性，可能对水解受损的突变酶的进一步反应具有抗性。对于所有酶，均获得了不溶性聚合物材料，其中DP10为主要成分，但也获得了其他不同DP的COS，这限制了生产具有确定DP的寡聚物/聚合物的实际应用。突变酶的残余水解酶活性与溶解度/沉淀动力学之间的平衡仍会导致新形成产物上的水解和/或转糖基化反应。从所选酶中，嗜热栖热菌ChiA D1022A突变体给出了最佳结果，以45%的产率（w/w）形成了不溶性聚合物，且含有约55%的目标DP10产物。
