Characterization of XynC from Bacillus subtilis subsp. subtilis strain 168 and analysis of its role in depolymerization of glucuronoxylan

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.

FIG. 1.

Optimization of XynC was performed using standard reaction conditions described in Materials and Methods. Buffer and pH conditions were optimized and were applied for determination of the optimal reaction temperature and half-life analyses. These results were used to define the reaction conditions for kinetic analysis. (A) Buffer and pH optimization using 50 mM buffers with a pH between 5.0 and 6.5. Diamonds, potassium phosphate; squares, sodium acetate. (B) Temperature optimization using 50 mM sodium acetate, pH 6.0. (C) Half-life analysis determined by preincubating XynC at the specified temperatures and measuring remaining activity over time. Data are presented as the half-lives obtained from the linear regression of inactivation at each temperature. (D) Lineweaver-Burk kinetic analysis was based on a reaction velocity versus substrate concentration data set which was fit to a logarithmic equation.

FIG. 2.

MALDI-TOF MS analysis of the YM-3 filtrate (A) and YM-3 retentate (B) resulting from 3-kDa ultrafiltration of an SG MeGAX_n XynC digest. Peak m/z values were tabulated and show that each cluster of peaks is composed of various single- and double-salt adducts which differ from the previous cluster by a single xylose residue. Each chemical species is composed of the designated number of xylose residues containing a single MeGA residue. Complements of different sodium and/or potassium adducts comprise designated clusters.

FIG. 3.

¹H NMR of SG MeGAX_n 3-kDa filtrate revealing the general action of XynC hydrolysis of MeGAX_n and the predicted limit product of XynC MeGAX_n digestion. Integrated intensity values for specific shift positions have been used to determine the product of an XynC digestion, establishing that there is a single MeGA substitution for every reducing terminal xylose and every nonreducing terminal xylose and that this substitution is penultimate to the reducing terminal xylose. (A) Shift assignments are labeled as follows: α,β-U1, 4-O-methylglucuronic acid carbon one hydrogen; U5, 4-O-methylglucuronic acid carbon five hydrogen; α,βr-X1, reducing terminal xylose carbon one hydrogen; nr-X5, nonreducing terminal xylose carbon five hydrogen; int-X5, internal xylose carbon five hydrogen. (B) Limit product generated by XynC-catalyzed hydrolysis of MeGAX_n. X, xylose; MeGA, 4-O-methylglucuronic acid; n, some number of β-1,4-linked xylose residues.

FIG. 4.

Identification of products generated by XynA (GH 11) and XynC (GH 5) secreted by B. subtilis 168. Spent medium from a mid-log-phase culture of B. subtilis 168 was concentrated by YM-3 filtration to provide the BSC fraction. This was fractionated using a BioGel P-60 column to provide fractions A and B, which were used to digest SG MeGAX_n and identify the xylanase hydrolysis pattern by TLC. SG MeGAX_n and an SG MeGAX_n digested with recombinantly expressed XynC were used as controls. Lanes: 1, SG MeGAX_n; 2, SG MeGAX_n digestion with fraction A; 3, SG MeGAX_n digestion with recombinant XynC; 4, MeGAX₁ to MeGAX₄ aldouronate standards; 5, xylooligomer standards with one to four xyloses; 6, SG MeGAX_n digestion with fraction B; 7, SG MeGAX_n digestion with BSC.

FIG. 5.

Regulation of expression of xynA and xynC genes in early- to mid-exponential-phase growth cultures of B. subtilis 168 with different sugars as the substrate, measured by using Q-RT-PCR. G, glucose; A, arabinose; B, birch wood MeGAX_n; S, sweetgum wood MeGAX_n; 1, xynA; 2, xynC.

FIG. 6.

Limit aldouronates expected from an SG MeGAX_n digestion with a GH 11 xylanase and a GH 5 xylanase cosecreted in the growth medium of B. subtilis 168. (A) MeGAX₄ with a MeGA substitution penultimate to the nonreducing terminal xylose, the smallest aldouronate product resulting from a GH 11 hydrolysis of MeGAX_n (4). (B) The predicted hydrolysis limit product of a GH 5 xylanase as presented in this publication, having a single MeGA substitution penultimate to the reducing terminal xylose. (C) MeGAX₃ with a MeGA substitution on the second of three xylose residues positioned penultimate to the reducing and nonreducing ends.