# Discovery and In Silico Characterization of Anatolian Water Buffalo Rumen-Derived Bacterial Thermostable Xylanases: A Sequence-Based Metagenomic Approach

## TL;DR

This study used metagenomic sequencing to identify and analyze thermostable xylanases from the rumen of Anatolian water buffalos, offering insights for industrial applications.

## Contribution

The study introduces novel thermostable xylanases from Anatolian water buffalo rumen and provides in-depth computational insights into their structure and function.

## Key findings

- Three xylanases (AWBRMetXyn5, AWBRMetXyn10, and AWBRMetXyn19) showed high thermostability with Tm scores above 70°C.
- Structural analysis revealed higher salt bridges and hydrophobic interactions in the xylanases compared to a reference thermophilic xylanase.
- Key amino acid variations and loop regions were identified as potential targets for improving thermostability and catalytic efficiency.

## Abstract

This study involved shotgun sequencing of rumen metagenomes
from three Anatolian water buffalos, an exploration of the relationship
between microbial flora and xylanases, and in silico analyses of thermostable xylanases, focusing on their sequence,
structure, and dynamic properties. For this purpose, the rumen metagenome
of three Anatolian water buffalos was sequenced and bioinformatically
analyzed to determine microbial diversity and full-length xylanases.
Analyses of BLAST, biophysicochemical characteristics, phylogenetic
tree, and multiple sequence alignment were performed with Blastp,
ProtParam, MEGA11 software, and Clustal Omega, respectively. Three-dimensional
homology models of three xylanases (AWBRMetXyn5, AWBRMetXyn10, and
AWBRMetXyn19) were constructed by SWISS-MODEL and validated by ProSA,
ProCheck, and Verify3D. Also, their 3D models were structurally analyzed
by PyMOL, BANΔIT, thermostability predictor, What If, and Protein
Interaction Calculator (PIC) software. Protein–ligand interactions
were examined by docking and MD simulation. Shotgun sequence and Blastp
analyses showed that Clostridium (Clostridiales bacterial
order), Ruminococcus (Oscillospiraceae bacterial
family), Prevotella (Bacteroidales bacterial order),
and Butyrivibrio (Lachnospiraceae bacterial family)
were found as dominant potential xylanase-producer genera in three
rumen samples. Furthermore, the biophysicochemical analysis indicated
that three xylanases exhibited an aliphatic index above 80, an instability
index below 40, and melting temperatures (Tm) surpassing 65 °C. Phylogenetic analysis placed three xylanases
within the GH10 family, clustering them with thermophilic xylanases,
while homology modeling identified the optimal template as a xylanase
from a thermophilic bacterium. The structural analysis indicated that
three xylanases possessed the number of salt bridges, hydrophobic
interactions, and Tm score higher than
50, 165, and 70 °C, respectively; however, the reference thermophilic
XynAS9 had 43, 145, and 54.41 °C, respectively. BANΔIT
analysis revealed that three xylanases exhibited lower B′-factor values in the β3-α1 loop/short-helix
at the N-terminal site compared to the reference thermophilic XynAS9.
In contrast, six residues (G79, M123, D150, T199, A329, and G377)
possessed higher B′-factor values in AWBRMetXyn5
and their aligned positions in AWBRMetXyn10 and AWBRMetXyn19, relative
to XynAS9 including Gln, Glu, Ile, Lys, Ser, and Val at these positions,
respectively. MD simulation results showed that the β9-η5
loop including catalytic nucleophile glutamic acid in the RMSF plot
of three xylanases had a higher fluctuation than the aligned region
in XynAS9. The distance analysis from the MD simulation showed that
the nucleophile residue in AWBRMetXyn5 and AWBRMetXyn10 remained closer
to the ligand throughout the simulation compared with XynAS9 and AWBRMetXyn19.
The most notable difference between AWBRMetXyn5 and AWBRMetXyn10 was
the increased amino acid fluctuations in two specific regions, the
η3
short-helix and the η3-α3 loop, despite a minimal sequence
difference of only 1.24%, which included three key amino acid variations
(N345, N396, and T397 in AWBRMetXyn5; D345, K396, and A397 in AWBRMetXyn10).
Thus, this study provided computational insights into xylanase function
and thermostability, which could inform future protein engineering
efforts. Additionally, three xylanases, especially AWBRMetXyn5, are
promising candidates for various high-temperature industrial applications.
In a forthcoming study, three xylanases will be experimentally characterized
and considered for potential industrial applications. In addition,
the amino acid substitutions (G79Q, M123E, D150I, T199K, A329S, and
G377V) and the residues in the β3-α1 loop will be targeted
for thermostability improvement of AWBRMetXyn5. The amino acids (N345,
N396, and T397) and the residues on the β9-η5 loop, η3
short-helix, and η3-α3 loop will also be focused on development
of the catalytic efficiency.