Biodiversité et Biotechnologie Fongiques, Aix-Marseille Université, INRAE, UMR1163 Marseille, France.
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Ecole Nationale d'Ingénieurs de Sfax, Université de Sfax, Sfax 3029, Tunisia.
Int J Mol Sci. 2020 Nov 9;21(21):8402. doi: 10.3390/ijms21218402.
Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus . The laccase-encoding gene Lac2 from was cloned for heterologous expression in D15#26 for protein production in the extracellular medium of around 30 mg L. The extracellular recombinant enzyme Lac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%. Lac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of Lac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate, Lac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min. Lac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt.
只有少数研究考察了海洋来源真菌及其酶如何适应盐度和植物生物质降解。这项工作涉及从海洋来源真菌的转录组中鉴定出一种氧化酶的生产和特性。 从 中克隆了编码漆酶的基因 Lac2 ，用于在 D15#26 中异源表达，以在约 30 mg L 的细胞外培养基中生产蛋白质。在超滤、阴离子交换层析和分子筛层析三个步骤的方案中，成功地生产和纯化了重组酶 Lac2，最终回收率为 24%。通过理化性质、动力学参数和氧化多种酚类底物的能力对 Lac2 进行了表征。我们还研究了它在有和没有海水盐存在下的活性。Lac2 的分子量约为 75 kDa，与大多数子囊菌真菌漆酶的分子量一致。以愈创木酚嗪为底物，Lac2 在 pH 6 时表现出最佳活性，在 50°C 孵育 180 分钟后保留近 100%的活性。在 5%wt/vol 的海水中，Lac2 的活性超过 50%。
