Exploring the soil mangrove diversity for new fungal DyPs

We recently investigated the structural organization of fungi in mangrove sediments of New Caledonia. In the present work, the objective was to extend this research by studying the functional diversity of the same mangrove sediments, i.e. getting data of fungal enzymes involved in the decomposition of the dead biomass accumulated in the sediments, together with their complete biochemical characterization. The diversity of expressed genes en-coding fungal DyPs was investigated in surface and deeper mangrove sediments beneath Avicennia marina and Rhizophora stylosa trees during the wet and dry seasons using a functional metabarcoding approach. For in-stance, DyP diversity was demonstrated to be higher in surface beneath the R. stylosa pristine area during the wet season, and Operational Functional Unit (OFU) 1 was detected in all sediment samples representing 51 to 100% of the total number sequences in 70% of the samples. To study this functional biodiversity, a dye-decolorizing peroxidase-encoding (DyP) gene corresponding to OFU1 was selected and cloned in Pichia pastoris for het-erologous expression. The recombinant enzyme, DyP1, was characterized concerning its physicochemical properties and ability to oxidize diverse phenolic substrates, together with its potential in decolorizing textile dyes. The optimal pH of DyP1 differed in relation to the substrates tested, and DyP1 was more active in acidic pH and stable in a rather large pH range. The enzyme has shown a very stable activity at temperatures up to 50 ?C, retaining 60 to 80% activity after 180 min of incubation at temperatures ranging from 30 to 70 ?C. A wide substrate specificity was observed with the lowest Km and highest catalytic efficiency values obtained against ABTS and Reactive blue 19, respectively. The industrial dyes Acid Black, Disperse blue 79 and Reactive Black 5 were decolorized with different percentages. Its activity related to salt was also studied and compared to other enzymes originating from marine-derived fungi.

本团队近期对新喀里多尼亚红树林沉积物中的真菌群落结构组成开展了研究。本研究旨在延伸此前的工作，对同一红树林沉积物的功能多样性展开探究：即获取参与沉积物中累积死亡生物质降解的真菌酶类相关数据，并完成其完整的生化特性表征。本研究采用功能元条形码（functional metabarcoding）技术，在湿季与旱季下，对红海榄（Rhizophora stylosa）和白骨壤（Avicennia marina）林下表层及深层红树林沉积物中，编码真菌染料脱色过氧化物酶（DyP, dye-decolorizing peroxidase）的表达基因多样性进行了分析。例如，研究发现湿季下红海榄原生林地表层沉积物中的DyP多样性更高；功能操作单元（OFU, Operational Functional Unit）1在所有沉积物样本中均被检出，在70%的样本中其序列数占总序列数的51%至100%。为深入探究该功能生物多样性，本研究选取了对应OFU1的染料脱色过氧化物酶编码基因（DyP），在毕赤酵母（Pichia pastoris）中进行克隆以实现异源表达。随后对重组酶DyP1进行了表征，涵盖其理化特性、氧化多种酚类底物的能力，以及对纺织染料的脱色潜力。DyP1的最适pH因所检测底物而异，其在酸性pH条件下活性更强，且在较宽的pH范围内均可保持稳定。该酶在最高50℃的条件下仍可保持稳定的活性；在30℃至70℃的温度下孵育180分钟后，其活性仍可保留60%至80%。DyP1展现出较广的底物特异性，其对ABTS的米氏常数（Km）最低，对活性蓝19（Reactive blue 19）的催化效率最高。酸性黑、分散蓝79与活性黑5等工业染料均可被该酶脱色，且脱色率各不相同。本研究还探究了该酶的盐浓度依赖活性，并将其与其他海洋来源真菌所产酶类进行了对比。