Dataset Information

Proteomic Analysis of the Xanthan-Degrading Pathway of Microbacterium sp. XT11.

ABSTRACT: Xanthan, a highly stable polysaccharide which is not easily degraded by most microorganisms, contains a cellulosic backbone with trisaccharide side chains composed of mannosyl-glucuronyl-mannose attached ?-1,3 to alternating glucosyl residues. Different digestion strategies were first applied to demonstrate the complexity about the proteomes of Microbacterium sp. XT11 in xanthan medium and glucose medium. Significantly up-regulated proteins induced by xanthan were screened out by the label-free quantitation of the proteomes of Microbacterium sp. XT11 in xanthan medium and glucose medium. Consequently, 2746 and 2878 proteins were identified in proteomes of Microbacterium sp. XT11 in xanthan medium and glucose medium individually, which represent 80.6 and 84.4% of total protein dataset predicted to be expressed by the gene. In the list of 430 induced proteins containing the proteins specifically expressed or up-regulated in xanthan medium, 19 proteins involved in carbohydrate-active enzymes database and 38 proteins annotated with transporter activity were critical in the degrading pathway of xanthan. Four CAZymes (GH3, GH38, GH9, and PL8) and one ABC transporter (LX1-1GL001097) were verified with quantitative real-time polymerase chain reaction. Four CAZymes (GH3, GH38, GH9, and PL8) were further verified with the enzyme assay. This study suggests a xanthan-degrading pathway in Microbacterium sp. XT11, and other potential xanthan degradation-related proteins still need further investigation.

SUBMITTER: Sun Z

PROVIDER: S-EPMC6868878 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Proteomic Analysis of the Xanthan-Degrading Pathway of <i>Microbacterium</i> sp. XT11.

Sun Zhen Z Liu Huixue H Wang Xueyan X Yang Fan F Li Xianzhen X

ACS omega 20191106 21

Xanthan, a highly stable polysaccharide which is not easily degraded by most microorganisms, contains a cellulosic backbone with trisaccharide side chains composed of mannosyl-glucuronyl-mannose attached α-1,3 to alternating glucosyl residues. Different digestion strategies were first applied to demonstrate the complexity about the proteomes of <i>Microbacterium</i> sp. XT11 in xanthan medium and glucose medium. Significantly up-regulated proteins induced by xanthan were screened out by the labe ...[more]

PMID: 31763532

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Project description:Under general aqueous conditions, xanthan appears in an ordered conformation, which makes its backbone largely resistant to degradation by known cellulases. Therefore, the xanthan degradation mechanism is still unclear because of the lack of an efficient hydrolase. Here, we report the catalytic properties of MiXen, a xanthan-degrading enzyme identified from the genus Microbacterium MiXen is a 952-amino-acid protein that is unique to strain XT11. Both the sequence and structural features suggested that MiXen belongs to a new branch of the GH9 family and has a multimodular structure in which a catalytic (?/?)6 barrel is flanked by an N-terminal Ig-like domain and by a C-terminal domain that has very few homologues in sequence databases and functions as a carbohydrate-binding module (CBM). Based on circular dichroism, shear-dependent viscosity, and reducing sugar and gel permeation chromatography analysis, we demonstrated that recombinant MiXen efficiently and randomly cleaved glucosidic bonds within the highly ordered xanthan substrate. A MiXen mutant free of the C-terminal CBM domain partially lost its xanthan-hydrolyzing ability because of decreased affinity toward xanthan, indicating the CBM domain assisted MiXen in hydrolyzing highly ordered xanthan via recognizing and binding to the substrate. Furthermore, side chain substituents and the terminal mannosyl residue significantly influenced the activity of MiXen via the formation of barriers to enzymolysis. Overall, the results of this study provide insight into the hydrolysis mechanism and enzymatic properties of a novel endotype xanthanase that will benefit future applications.IMPORTANCE This work characterized a novel endotype xanthanase, MiXen, and elucidated that the C-terminal carbohydrate-binding module of MiXen could drastically enhance the hydrolysis activity of the enzyme toward highly ordered xanthan. Both the sequence and structural analysis demonstrated that the catalytic domain and carbohydrate-binding module of MiXen belong to the novel branch of the GH9 family and CBMs, respectively. This xanthan cleaver can help further reveal the enzymolysis mechanism of xanthan and provide an efficient tool for the production of molecular modified xanthan with new physicochemical and physiological functions.

Project description:BackgroundLignin is a major component of plant biomass and is recalcitrant to degradation due to its complex and heterogeneous aromatic structure. The biomass-based research mainly focuses on polysaccharides component of biomass and lignin is discarded as waste with very limited usage. The sustainability and success of plant polysaccharide-based biorefinery can be possible if lignin is utilized in improved ways and with minimal waste generation. Discovering new microbial strains and understanding their enzyme system for lignin degradation are necessary for its conversion into fuel and chemicals. The Pandoraea sp. ISTKB was previously characterized for lignin degradation and successfully applied for pretreatment of sugarcane bagasse and polyhydroxyalkanoate (PHA) production. In this study, genomic analysis and proteomics on aromatic polymer kraft lignin and vanillic acid are performed to find the important enzymes for polymer utilization.ResultsGenomic analysis of Pandoraea sp. ISTKB revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin degradation and PHA production. We also applied label-free quantitative proteomic approach to identify the expression profile on monoaromatic compound vanillic acid (VA) and polyaromatic kraft lignin (KL). Genomic and proteomic analysis simultaneously discovered Dyp-type peroxidase, peroxidases, glycolate oxidase, aldehyde oxidase, GMC oxidoreductase, laccases, quinone oxidoreductase, dioxygenases, monooxygenases, glutathione-dependent etherases, dehydrogenases, reductases, and methyltransferases and various other recently reported enzyme systems such as superoxide dismutases or catalase-peroxidase for lignin degradation. A strong stress response and detoxification mechanism was discovered. The two important gene clusters for lignin degradation and three PHA polymerase spanning gene clusters were identified and all the clusters were functionally active on KL-VA.ConclusionsThe unusual aerobic '-CoA'-mediated degradation pathway of phenylacetate and benzoate (reported only in 16 and 4-5% of total sequenced bacterial genomes), peroxidase-accessory enzyme system, and fenton chemistry based are the major pathways observed for lignin degradation. Both ortho and meta ring cleavage pathways for aromatic compound degradation were observed in expression profile. Genomic and proteomic approaches provided validation to this strain's robust machinery for the metabolism of recalcitrant compounds and PHA production and provide an opportunity to target important enzymes for lignin valorization in future.

Dataset Information

Proteomic Analysis of the Xanthan-Degrading Pathway of Microbacterium sp. XT11.

Publications

Proteomic Analysis of the Xanthan-Degrading Pathway of <i>Microbacterium</i> sp. XT11.

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