Complete genome sequence of a xanthan-degrading Microbacterium sp. strain XT11 with the potential for xantho-oligosaccharides production

Yang, Fan; Li, Lili; Si, Yang; Yang, Ming; Guo, Xiaoyu; Hou, Yali; Chen, Xiaoyi; Li, Xianzhen

doi:10.1016/j.jbiotec.2016.02.005

Cited by 10 publications

(22 citation statements)

References 10 publications

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“…XT11. 18 Moreover, the structures of xanthan depolymerization products were determined by Nankai et al 14 Therefore, we suggested a xanthan-degrading pathway in Microbacterium sp. XT11 according to the abovementioned results of proteomic analysis.…”

Section: Resultsmentioning

confidence: 77%

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

et al. 2019

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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 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.

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Section: Resultsmentioning

confidence: 77%

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

et al. 2019

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“…Previously, an aerobic Gram-positive bacterium, Microbacterium sp. XT11, with the ability to degrade xanthan, was isolated from a soil sample collected from the Dalian Botanical Garden in China (16), and the complete genome sequence of strain XT11 was determined and analyzed (17). Within the genomic sequence of Microbacterium sp.…”

Section: Resultsmentioning

confidence: 99%

“…A xanthanase in Microbacterium sp. strain XT11, MiXen, is presumed to be a key xanthan-degrading enzyme because (i) the gene encoding MiXen is located in a xanthan-degrading gene cluster in strain XT11 (17) and (ii) sequence and structural features suggest that MiXen belongs to the glycoside hydrolase family 9 (Fig. 1), most members of which have been characterized as cellulases.…”

Section: Discussionmentioning

confidence: 99%

Novel Endotype Xanthanase from Xanthan-Degrading Microbacterium sp. Strain XT11

Yang

Sun

et al. 2019

Appl Environ Microbiol

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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 (α/α)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.

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“…Recent whole-genome analysis indicates that some xanthan-utilizing bacteria, exemplified by Microbacterium sp. strain XT11, encode these enzymes and requisite sugar transporters via a single, transcriptionally coregulated gene cluster 15 .…”

Section: Introductionmentioning

confidence: 99%

“…Commensurate with the structural complexity of xanthan, complete saccharification involves sequential action of an extracellular xanthan lyase, an extracellular endo -β-glucanase, and three intracellular exo -glycosidases: β-glucosidase, β-glucuronidase, and α-mannosidase. ,− Recent whole-genome analysis indicates that some xanthan-utilizing bacteria, exemplified by Microbacterium sp. strain XT11, encode these enzymes and requisite sugar transporters via a single transcriptionally coregulated gene cluster …”

Section: Introductionmentioning

confidence: 99%

Structural Dynamics and Catalytic Properties of a Multimodular Xanthanase

et al. 2018

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The precise catalytic strategies used for the breakdown of the complex bacterial polysaccharide xanthan, an increasingly frequent component of processed human foodstuffs, have remained a mystery. Here we present the characterization of an endo-xanthanase from Paenibacillus sp. 62047. We show that it is a CAZy family 9 glycoside hydrolase (GH9) responsible for the hydrolysis of the xanthan backbone, capable of generating tetrameric xanthan oligosaccharides from polysaccharide lyase family 8 (PL8) xanthan lyase-treated xanthan. 3-D structure determination reveals a complex multi-modular enzyme in which a catalytic (α/α)6 barrel is flanked by an N-terminal "immunoglobulin-like" (Ig-like) domain (frequently found in GH9 enzymes) and by four additional C-terminal all β-sheet domains which have very few homologs in sequence databases and, at least, one of which functions as a new xanthan-binding domain, now termed CBM84. The solution phase conformation and dynamics of the enzyme in the native calcium-bound state and in the absence of calcium were probed experimentally by hydrogen/deuterium exchange mass spectrometry. Measured conformational dynamics were used to guide the protein engineering of enzyme variants with increased stability in the absence of calcium; a property of interest for the potential use of the enzyme in cleaning detergents. The ability of hydrogen/deuterium exchange mass spectrometry to pinpoint dynamic regions of a protein under stress (e.g. removal of calcium ions) makes this technology a strong tool for improving protein catalyst properties by informed engineering.

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Complete genome sequence of a xanthan-degrading Microbacterium sp. strain XT11 with the potential for xantho-oligosaccharides production

Cited by 10 publications

References 10 publications

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

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

Novel Endotype Xanthanase from Xanthan-Degrading Microbacterium sp. Strain XT11

Structural Dynamics and Catalytic Properties of a Multimodular Xanthanase

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