Affiliation:
1. Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida
Abstract
ABSTRACT
Secretion of xylanase activities by
Bacillus subtilis
168 supports the development of this well-defined genetic system for conversion of methylglucuronoxylan (MeGAX
n
[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 MeGAX
n
, 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 MeGAX
n
as the substrate, the specific activity increased with increasing methylglucuronosyl substitutions on the β-1,4-xylan chain. With MeGAX
n
from sweetgum as a preferred substrate, XynC exhibited a
V
max
of 59.9 units/mg XynC, a
K
m
of 1.63 mg MeGAX
n
/ml, and a
k
cat
of 2,635/minute at pH 6.0 and 37°C. Matrix-assisted laser desorption ionization—time of flight mass spectrometry and
1
H 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 MeGAX
n
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 MeGAX
n
.
B. subtilis
may serve as a model platform for development of gram-positive biocatalysts for conversion of lignocellulosic materials to renewable fuels and chemicals.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
106 articles.
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