Characterization of XynC from Bacillus Subtilis Subsp. Subtilis Strain 168 and Analysis of Its Role in Depolymerization of Glucuronoxylan

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 2006 Oct 10

PMID 17028274

Citations 41

Authors

Franz J St John

John D Rice

James F Preston

Affiliations

Soon will be listed here.

Abstract

Secretion of xylanase activities by Bacillus subtilis 168 supports the development of this well-defined genetic system for conversion of methylglucuronoxylan (MeGAXn [where n represents the number of xylose residues]) in the hemicellulose component of lignocellulosics to biobased products. In addition to the characterized glycosyl hydrolase family 11 (GH 11) endoxylanase designated XynA, B. subtilis 168 secretes a second endoxylanase as the translated product of the ynfF gene. This sequence shows remarkable homology to the GH 5 endoxylanase secreted by strains of Erwinia chrysanthemi. To determine its properties and potential role in the depolymerization of MeGAXn, the ynfF gene was cloned and overexpressed to provide an endoxylanase, designated XynC, which was characterized with respect to substrate preference, kinetic properties, and product formation. With different sources of MeGAXn as the substrate, the specific activity increased with increasing methylglucuronosyl substitutions on the beta-1,4-xylan chain. With MeGAXn from sweetgum as a preferred substrate, XynC exhibited a Vmax of 59.9 units/mg XynC, a Km of 1.63 mg MeGAXn/ml, and a k(cat) of 2,635/minute at pH 6.0 and 37 degrees C. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and 1H nuclear magnetic resonance data revealed that each hydrolysis product has a single glucuronosyl substitution penultimate to the reducing terminal xylose. This detailed analysis of XynC from B. subtilis 168 defines the unique depolymerization process catalyzed by the GH 5 endoxylanases. Based upon product analysis, B. subtilis 168 secretes both XynA and XynC. Expression of xynA was subject to MeGAXn induction; xynC expression was constitutive with growth on different substrates. Translation and secretion of both GH 11 and GH 5 endoxylanases by the fully sequenced and genetically malleable B. subtilis 168 recommends this bacterium for the introduction of genes required for the complete utilization of products of the enzyme-catalyzed depolymerization of MeGAXn. B. subtilis may serve as a model platform for development of gram-positive biocatalysts for conversion of lignocellulosic materials to renewable fuels and chemicals.

Citing Articles

Use of xylosidase 3C from Segatella baroniae to discriminate xylan non-reducing terminus substitution characteristics.

St John F, Bynum L, Tauscheck D, Crooks C BMC Res Notes. 2024; 17(1):175.

PMID: 38915023 PMC: 11197168. DOI: 10.1186/s13104-024-06835-3.

Characterization of a novel GH30 non-specific endoxylanase AcXyn30B from Acetivibrio clariflavus.

Suchova K, Fathallah W, Puchart V Appl Microbiol Biotechnol. 2024; 108(1):312.

PMID: 38683242 PMC: 11058611. DOI: 10.1007/s00253-024-13155-w.

Xylanolytic Bacillus species for xylooligosaccharides production: a critical review.

Rashid R, Sohail M Bioresour Bioprocess. 2024; 8(1):16.

PMID: 38650226 PMC: 10991489. DOI: 10.1186/s40643-021-00369-3.

Carbohydrate-binding modules targeting branched polysaccharides: overcoming side-chain recalcitrance in a non-catalytic approach.

Liu J, Sun D, Zhu J, Liu C, Liu W Bioresour Bioprocess. 2024; 8(1):28.

PMID: 38650221 PMC: 10992016. DOI: 10.1186/s40643-021-00381-7.

Impact of xylan on field productivity and wood saccharification properties in aspen.

Derba-Maceluch M, Sivan P, Donev E, Gandla M, Yassin Z, Vaasan R Front Plant Sci. 2023; 14:1218302.

PMID: 37528966 PMC: 10389764. DOI: 10.3389/fpls.2023.1218302.

References

Ito Y, Tomita T, Roy N, Nakano A, Sugawara-Tomita N, Watanabe S . Cloning, expression, and cell surface localization of Paenibacillus sp. strain W-61 xylanase 5, a multidomain xylanase. Appl Environ Microbiol. 2003; 69(12):6969-78. PMC: 310030. DOI: 10.1128/AEM.69.12.6969-6978.2003. View

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

Bendtsen J, Nielsen H, von Heijne G, Brunak S . Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004; 340(4):783-95. DOI: 10.1016/j.jmb.2004.05.028. View

Clarke J, Rixon J, Ciruela A, Gilbert H, Hazlewood G . Family-10 and family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps. Appl Microbiol Biotechnol. 1997; 48(2):177-83. DOI: 10.1007/s002530051035. View

Kardosova A, Matulova M, Malovikova A . (4-O-Methyl-alpha-D-glucurono)-D-xylan from Rudbeckia fulgida, var. sullivantii (Boynton et Beadle). Carbohydr Res. 1998; 308(1-2):99-105. DOI: 10.1016/s0008-6215(98)00072-x. View

Sun Y, Cheng J . Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol. 2002; 83(1):1-11. DOI: 10.1016/s0960-8524(01)00212-7. View

Boraston A, Bolam D, Gilbert H, Davies G . Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J. 2004; 382(Pt 3):769-81. PMC: 1133952. DOI: 10.1042/BJ20040892. View

Nagy T, Nurizzo D, Davies G, Biely P, Lakey J, Bolam D . The alpha-glucuronidase, GlcA67A, of Cellvibrio japonicus utilizes the carboxylate and methyl groups of aldobiouronic acid as important substrate recognition determinants. J Biol Chem. 2003; 278(22):20286-92. DOI: 10.1074/jbc.M302205200. View

Jacob S, Allmansberger R, Gartner D, Hillen W . Catabolite repression of the operon for xylose utilization from Bacillus subtilis W23 is mediated at the level of transcription and depends on a cis site in the xylA reading frame. Mol Gen Genet. 1991; 229(2):189-96. DOI: 10.1007/BF00272155. View

10.

Lynd L, van Zyl W, McBride J, Laser M . Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol. 2005; 16(5):577-83. DOI: 10.1016/j.copbio.2005.08.009. View

11.

Khasin A, Alchanati I, Shoham Y . Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl Environ Microbiol. 1993; 59(6):1725-30. PMC: 182151. DOI: 10.1128/aem.59.6.1725-1730.1993. View

12.

BLUMENKRANTZ N, ASBOE-HANSEN G . New method for quantitative determination of uronic acids. Anal Biochem. 1973; 54(2):484-9. DOI: 10.1016/0003-2697(73)90377-1. View

13.

Bounias M . N-(1-naphthyl)ethylenediamine dihydrochloride as a new reagent for nanomole quantification of sugars on thin-layer plates by a mathematical calibration process. Anal Biochem. 1980; 106(2):291-5. DOI: 10.1016/0003-2697(80)90523-0. View

14.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

15.

Rozen S, Skaletsky H . Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 1999; 132:365-86. DOI: 10.1385/1-59259-192-2:365. View

16.

Tjalsma H, Antelmann H, Jongbloed J, Braun P, Darmon E, Dorenbos R . Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome. Microbiol Mol Biol Rev. 2004; 68(2):207-33. PMC: 419921. DOI: 10.1128/MMBR.68.2.207-233.2004. View

17.

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

18.

Kraus A, Hueck C, Gartner D, Hillen W . Catabolite repression of the Bacillus subtilis xyl operon involves a cis element functional in the context of an unrelated sequence, and glucose exerts additional xylR-dependent repression. J Bacteriol. 1994; 176(6):1738-45. PMC: 205262. DOI: 10.1128/jb.176.6.1738-1745.1994. View

19.

Pell G, Taylor E, Gloster T, Turkenburg J, Fontes C, Ferreira L . The mechanisms by which family 10 glycoside hydrolases bind decorated substrates. J Biol Chem. 2003; 279(10):9597-605. DOI: 10.1074/jbc.M312278200. View

20.

Pell G, Szabo L, Charnock S, Xie H, Gloster T, Davies G . Structural and biochemical analysis of Cellvibrio japonicus xylanase 10C: how variation in substrate-binding cleft influences the catalytic profile of family GH-10 xylanases. J Biol Chem. 2003; 279(12):11777-88. DOI: 10.1074/jbc.M311947200. View