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Characterization of Novel Pectinolytic Enzymes Derived from the Efficient Lignocellulose Degradation Microbiota.

ABSTRACT: Diverse pectinolytic enzymes are widely applied in the food, papermaking, and other industries, and they account for more than 25% of the global industrial enzyme demands. Efficient lignocellulose degradation microbiota are reservoirs of pectinolytic enzymes and other lignocellulose-degrading genes. Metagenomics has been widely used to discover new pectinolytic enzymes. Here, we used a metagenomic strategy to characterize pectinolytic genes from one efficient lignocellulose-degrading microbiota derived from pulp and paper wastewater treatment microbiota. A total of 23 predicted full-length GH28 and PL1 family pectinolytic genes were selectively cloned and expressed in Escherichia coli, and 5 of the expressed proteins had pectinolytic activities. Among them, the characterization of one pectinolytic enzyme, PW-pGH28-3, which has a 58.4% identity with an exo-polygalacturonase gene of Aquipluma nitroreducens, was further investigated. The optimal pH and optimal temperature of PW-pGH28-3 were 8.0 and 40 °C, respectively, and its pectinolytic activity at the optimal condition was 13.5 ± 1.1 U/mg protein. Bioinformatics analyses and structural modeling suggest that PW-pGH28-3 is a novel secretory exo-polygalacturonase, which is confirmed by its hydrolysates of polygalacturonic acid. The detection of PW-pGH28-3 and other pectinolytic genes showed that efficient lignocellulose degradation microbiota could provide potential efficient pectinolytic enzymes for industrial application. In the future, improving metagenomic screening efficiency would discover efficient lignocellulose-degrading enzymes and lead to the sustainable and green utilization of lignocellulose.

SUBMITTER: Miao Q

PROVIDER: S-EPMC9599418 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Characterization of Novel Pectinolytic Enzymes Derived from the Efficient Lignocellulose Degradation Microbiota.

Miao Qin Q Zhang Xiaoling X Wang Yitong Y Li Xiaoqi X Wang Zheng Z Tian Lingmin L Qu Lingbo L Wei Yongjun Y

Biomolecules 20220929 10

Diverse pectinolytic enzymes are widely applied in the food, papermaking, and other industries, and they account for more than 25% of the global industrial enzyme demands. Efficient lignocellulose degradation microbiota are reservoirs of pectinolytic enzymes and other lignocellulose-degrading genes. Metagenomics has been widely used to discover new pectinolytic enzymes. Here, we used a metagenomic strategy to characterize pectinolytic genes from one efficient lignocellulose-degrading microbiota ...[more]

PMID: 36291597

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Project description:Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with genes that could encode lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes: β-glucosidase, α-L-arabinofuranosidase, β-xylosidase, and endo-1,4-β-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation. Phylogenetic trees were created for each of these putative enzymes to depict genetic relatedness to known enzymes. Candidate genes were synthesized and cloned into plasmid expression vectors for inducible protein expression and secretion. The putative β-glucosidase fusion protein was efficiently secreted but did not permit Escherichia coli (E. coli) to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl β-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). The putative hemicellulose-degrading β-xylosidase and α-L-arabinofuranosidase enzymes also lacked in vitro enzyme activity, but the affinity purified endo-1,4-β-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7. At this optimal pH, KM, Vmax, and kcat were determined to be 32.005 ± 4.72 μM, 1.16x10-5 ± 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases.

Project description:BackgroundLignocellulose is the most abundant renewable carbon resource that can be used for biofuels and commodity chemicals production. The ability of complex microbial communities present in natural environments that are specialized in biomass deconstruction can be exploited to develop lignocellulose bioconversion processes. Termites are among the most abundant insects on earth and play an important role in lignocellulose decomposition. Although their digestive microbiome is recognized as a potential reservoir of microorganisms producing lignocellulolytic enzymes, the potential to enrich and maintain the lignocellulolytic activity of microbial consortia derived from termite gut useful for lignocellulose biorefinery has not been assessed. Here, we assessed the possibility of enriching a microbial consortium from termite gut and maintaining its lignocellulose degradation ability in controlled anaerobic bioreactors.ResultsWe enriched a termite gut-derived consortium able to transform lignocellulose into carboxylates under anaerobic conditions. To assess the impact of substrate natural microbiome on the enrichment and the maintenance of termite gut microbiome, the enrichment process was performed using both sterilized and non-sterilized straw. The enrichment process was carried out in bioreactors operating under industrially relevant aseptic conditions. Two termite gut-derived microbial consortia were obtained from Nasutitermes ephratae by sequential batch culture on raw wheat straw as the sole carbon source. Analysis of substrate loss, carboxylate production and microbial diversity showed that regardless of the substrate sterility, the diversity of communities selected by the enrichment process strongly changed compared to that observed in the termite gut. Nevertheless, the community obtained on sterile straw displayed higher lignocellulose degradation capacity; it showed a high xylanase activity and an initial preference for hemicellulose.ConclusionsThis study demonstrates that it is possible to enrich and maintain a microbial consortium derived from termite gut microbiome in controlled anaerobic bioreactors, producing useful carboxylates from raw biomass. Our results suggest that the microbial community is shaped both by the substrate and the conditions that prevail during enrichment. However, when aseptic conditions are applied, it is also affected by the biotic pressure exerted by microorganisms naturally present in the substrate and in the surrounding environment. Besides the efficient lignocellulolytic consortium enriched in this study, our results revealed high levels of xylanase activity that can now be further explored for enzyme identification and overexpression for biorefinery purposes.

Dataset Information

Characterization of Novel Pectinolytic Enzymes Derived from the Efficient Lignocellulose Degradation Microbiota.

Publications

Characterization of Novel Pectinolytic Enzymes Derived from the Efficient Lignocellulose Degradation Microbiota.

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