C. Vafiadi et al. / Process Biochemistry 45 (2010) 419–424
423
3.5. MS peptide sequencing
sequence, as predicted by using the NetNGlyc 1.0 server [29] and
by the experimental evidence where the endoglycosidase H
treatment produced the same mass polypeptides of both purified
xylanases (Fig. 1B).
The determination of the two xylanase genes from the recently
assembled genome of S. thermophile will allow us to design specific
primers for the expression of the expected recombinant enzymes.
The heterologous overexpression of the two xylanases from S.
thermophile will result in large amounts of recombinant xylanases
belonging in the GH families 10 and 11, which are enzymes with
many known industrial applications [30].
The purified S. thermophile xylanases, StXyn1 and StXyn2, were
Coomassie stained on a SDS-PAGE gel showing a band correspond-
ing to 24 kDa and 48 kDa (Fig. 1A), respectively, which were in-gel
digested with trypsin and the total digests were subjected to
nanoLC–ESI-MS/MS.
The peptide sequences found were used as query against the S.
thermophile genome sequence assembly v1.0 database (MASCOT
search), which was annotated by the JGI Genome Annotation
Pipeline. In case of StXyn1, three peptides were found to
correspond exactly to internal sequence of hypothetical xylanase
belonging to GH family 11 (Model ID 89603, scaffold_1:1795517–
1796301), while in case of StXyn2, only one peptide matched to
the internal sequence of hypothetical xylanase belonging to
family 10 (Model ID 112050, scaffold_6:32846–34155) from the
genome of S. thermophile (Table 2). The results presented by the
MS identification were strong evidence that StXyn1 and StXyn2
belong to GH11 and GH10 families [1], while correspond to the
gene models 89603 and 112050 of the S. thermophile database
v1.0, respectively. The nominal mass of the translated mature
hypothetical xylanase StXyn1 (Model ID 89603) and its calculated
pI value were found 23,125 Da and 6.71, respectively, as estimated
using the ProtParam tool of ExPASy [21]. The StXyn1 mature
protein was estimated by the exclusion of the secretion signal
peptide (MVSLKSLLLAAAATLTAVTA, 20aa) based upon the pre-
diction using SignalP, which is a web-based program [22]. These
putative and experimentally physicochemical values found are
favorably compared to the experimentally determined ones of the
purified StXyn1 (24 kDa, pI 8.7; Fig. 1). On the other hand, using
the aforementioned bioinformatics tools, the nominal mass of the
translated mature hypothetical xylanase StXyn2 (Model ID
112050; excluding secretion signal MHSKAFLAALLAPAVSG,
17aa) and its calculated pI value were found 42,859 Da and
5.52, respectively, which differ considerably from experimentally
determined values, especially in case of the MW of the purified
StXyn2 (48 kDa, pI 8.0; Fig. 1). The hypothetical StXyn2 xylanase
contains a family 1 Carbohydrate Binding Module (CBM) situated
in the C-terminal of the putative protein, as estimated using the
InterProScan tool [23]. In order to test the cellulose-binding
ability of StXyn2, binding of xylanase on Avicel cellulose was
4. Conclusion
The above study represents a direct application of LC–MS/MS
technology for the gene identification and classification of endo-
1,4-b-xylanases in the continually updated Carbohydrate-Active
enzymes (CAZy) database. The thermophilic xylanases from S.
thermophile may be suitable for industrial applications such as
hydrolysis of lignocellulosic biomass where thermostability would
enable to decrease the costs of the hydrolysis step and allow more
flexible process configurations; e.g. at higher temperature ranges
and at higher dry matter consistencies.
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