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Anand D, Nasim J, Yadav S, Yadav D. Bioinformatics Insights Into Microbial Xylanase Protein Sequences. Biosci Biotech Res Asia 2018;15(2).
Manuscript received on : 18 May 2018
Manuscript accepted on : 25 June 2018
Published online on:  20180627
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Bioinformatics Insights Into Microbial Xylanase Protein Sequences

Deepsikha Anand, Jeya Nasim, Sangeeta Yadav and Dinesh Yadav

Department of Biotechnology, DDU Gorakhpur University, Gorakhpur (U.P) 273009, India.

Corresponding Author E-mail: dinesh_yad@rediffmail.com

DOI : http://dx.doi.org/10.13005/bbra/2631

ABSTRACT: Microbial xylanases represents an industrially important group of enzymes associated with hydrolysis of xylan, a major hemicellulosic component of plant cell walls. A total of 122 protein sequences comprising of 58 fungal, 25 bacterial, 19actinomycetes and 20 yeasts xylanaseswere retrieved from NCBI, GenBank databases. These sequences were in-silico characterized for homology,sequence alignment, phylogenetic tree construction, motif assessment and physio-chemical attributes. The amino acid residues ranged from 188 to 362, molecular weights were in the range of 20.3 to 39.7 kDa and pI ranged from 3.93 to 9.69. The aliphatic index revealed comparatively less thermostability and negative GRAVY indicated that xylanasesarehydrophilicirrespective of the source organisms.Several conserved amino acid residues associated with catalytic domain of the enzyme were observed while different microbial sources also revealed few conserved amino acid residues. The comprehensive phylogenetic tree indicatedsevenorganismsspecific,distinct major clusters,designated as A, B, C, D, E, F and G. The MEME based analysis of 10 motifs indicated predominance of motifs specific to GH11 family and one of the motif designated as motif 3 with sequence GTVTSDGGTYDIYTTTRTNAP was found to be present in most of the xylanases irrespective of the sources.Sequence analysis of microbial xylanases provides an opportunity to develop strategies for molecular cloning and expression of xylanase genes and also foridentifying sites for genetic manipulation for developing novel xylanases with desired features as per industrial needs.

KEYWORDS: Bioinformatics; Multiple sequence Alignment; Source Organisms; Xylanases; Phylogenetic Tree

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Introduction

Plant cell wall comprises of three major constituent namely cellulose, hemicelluloses and lignin.Xylan is the major hemicellulosic component and is the second most abundant polysaccharides in nature.Hemicellulose is a branched heteropolymer consisting of pentose and hexose sugars with xylose being most abundant1 (Kumar et al., 2008). A repertoire of enzymes including endo-xylanase (endo-1,4-β-xylanase; E.C.3.2.1.8), β-xylosidase (xylan-1,4-β-xylosidase; E.C.3.2.1.37), α-glucosiduronase (E.C.3.2.1.139),α-arabinofuranosidase(E.C.3.2.1.55) and acetylxylan esterase, (E.C.3.1.1.72)are associated with complete hydrolysis of hemicellulose2(Juturu and Wu, 2012). The endo-xylanase and β-xylosidase are key enzymes associated with hydrolysis of xylan and are collectively referred as xylanases.

Xylanasesbelongs to enzyme class of glycoside hydrolases (GH), which are classified into several families based on amino acid sequences. Xylanases is predominantly represented in two families GH10 and GH113-5 (Paes et al., 2012;Lafond et al., 2014; Chakdar et al., 2016).Xylanases have been reported from diverse microbial sources namely fungi, bacteria,actinomycetes and yeast and have been reviewed extensively over the years2,6-10 (Beg et al., 2001; Collins et al., 2005; Nair et al., 2008; Juturu and Wu, 2012; Juturu and Wu, 2014; Walia et al., 2017).Xylanasesrepresents industrially important group of enzymes with diverse applications like pulp and paper bleaching, fruit juice clarification, bioethanol production, bioconversion etc.2,11-14.(Shatalov et al., 2008; Valls et al., 2010; Juturu and Wu, 2012; Singh et al., 2013; Walia et al., 2015).

Several bioinformatics studies have been done on various xylanases. In-silico analysis of structural attributes ofcommercially important xylanases from diverse sources structural has beenreported15(Arora et al., 2009).Attempts have been made to study the structural dynamics changes of the Trichodermalongibrachiatumxylanase upon binding with xylohexaose and xylan ligands16 (Uzuner et al., 2010). In-silico structural prediction of Bacillus brevisxylanase and its comparative assessment with few bacterial and fungal xylanases has been reported recently17(Mathur et al., 2015).Efforts have been made to analyze several plant cell wall degrading enzymes (PCWDEs) including xylanses and polygalacturonasesofFusariumvirguliformeusing bioinformatics tools to develop fungal resistant soyabean18(Chang et al., 2016). Homology modeling of xylanase from Aspergillusfumigatus R1 isolate to get an insight into three dimensional structurehas been attempted19 (Deshmukh et al., 2016).

This manuscript reports in-silico characterization of xylanase protein sequences retrieved from NCBI representing diverse microbial sources namely fungi, bacteria, actinomycetes and yeast. Bioinformatics assessment of these sequences for homology,sequence alignment, physio-chemical attributes, motif assessment and phylogenetic tree construction isreported.The bioinformatics driven characterization of available sequences of microbial xylanases could be utilized for developing appropriate strategies for molecular cloning and expression of xylanasegenes.Further, the sequence-structure-function relationship could be established from in-silico studies and novel xylanases could be derived using state-of-the art technologies either metagenomics or directed evolution approaches.

Materials and Methods

Database Search and Sequence Retrieval

Xylanase protein sequences representing different microbial sources were retrieved from GenBank, NCBI (http//www.ncbi.nlm.nih.gov/).The sequences retrieved were saved in FASTA format and truncated proteins were discarded. The major groups as source organisms represents  fungi, bacteria, actinomycetes and yeast and the all the sequences of xylanases belongs to GH11 family.

Physio-Chemical Attributes

The physio-chemical attributes namely molecular weight, theoretical pI, aliphatic index, instability index, Grand Average of Hydropathicity (GRAVY) were analyzed by ProtParam tool (http://web.expasy.org/protparam/).20(Gasteiger et al.,2005).

Multiple sequence Alignment and Phylogenetic Analysis

The protein alignment of full length amino acid sequences of xylanase were performed by CLUSTAL X version 2.121(Larkin et al., 2007). Phylogenetic tree was constructed by NJ method using the MEGA 7.0 program22(Kumar et al., 2016) based on protein sequences.

Identification of conserved motifs

The protein sequences of xylanase were analyzed by Multiple EM for Motif Elicitation(MEME)program version 4.12.0 (http://meme.nbcr.net/meme/)23(Timothy et al.,2009).The maximum number of motifswereset as 10.The minimum width of 6 and maximum width of 50 amino acids was set along with other factors as default values.

Results and Discussion

Physio-chemical characterization of xylanases

A total of the 122 xylanase protein sequences belonging to GH11 family representing 58 fungal, 25 bacterial, 19 actinomycetes and 20 yeastxylanaseswere retrieved from NCBI databases (Table-1).It has been reported that bacterial xylanases generally represent GH10 family though fungal xylanases predominantly belongs to GH11 family24(Liu et al., 2011).Theirphysio-chemical properties namely molecular weight, pI, instability index, aliphatic index, GRAVYwere analyzed using ProtParam tool (Table-1). Theamino acid residues ranged from 188-362 residueswhilemolecularweightwasin the range of 20.3-39.7 KDa. The Isoelectric point(pI)was in the range of 3.93-9.69.The molecular weight in the range of 8.5 to 85kDa and pI in the range of 4-10.3 has been reported for bacterial5(Chakdhar et al.,2015) and fungal xylanases25(Polizeli et al. 2005).

Table 1: List of xylanase protein sequences from different microbial sources with in-silicophysio-chemical attributes revealed by Protparam

S.No. Source Organism Accession No. Amino acid Mol.wt. pI Instability

index

Aliphatic

index

GRAVY
FUNGI
1 Aspergillusnomius XP_015411959 229 24.4 5.73 26.21 59.61 -0.33
2 Aspergillusnomius XP_015411268 221 23.8 4.55 22.18 60.9 -0.427
3 Aspergillusniger AAS46914 225 24.1 5.23 22.11 59.78 -0.396
4 Aspergillusniger AAS46913 211 22.5 4 19.86 59.62 -0.178
5 Aspergillusniger AAA99065 211 22.7 4.55 26.49 59.15 -0.18
6 Aspergillusniger AFK10491 225 24 5.2 21.06 58.53 -0.38
7 Aspergillusniger CAA03654 225 24 5.45 21.06 58.53 -0.384
8 Aspergillusniger ACN89393 225 24 5.2 21.06 58.53 -0.38
9 Aspergillusniger AAM95167 225 24.1 5.23 22.11 59.78 -0.396
10 Aspergillusniger ABA00146 225 24.1 5.23 22.11 59.78 -0.396
11 Aspergillusniger AGH29125 225 24.1 5.23 22.11 59.78 -0.396
12 Aspergillusflavus KOC12560 232 24.6 5.49 21.53 58.84 -0.291
13 Aspergillusfumigatus XP_751100 221 23.8 5.22 24.15 56.02 -0.422
14 Aspergillusfumigatus XP_748354 228 24.4 6.27 23.44 53.9 -0.361
15 Aspergillusfumigatus XP_748367 313 33 6.03 26.03 69.71 -0.093
16 Aspergillusnidulans CAA90074 221 23.5 4.54 32.04 54.71 -0.378
17 Aspergillusniger XP_001388522 225 24 5.2 21.06 58.53 -0.38
18 Aspergillusniger ACJ26382 225 24 5.2 21.06 58.53 -0.384
19 Aspergillusniger ACA24724 225 24 5.44 20.72 59.82 -0.346
20 Aspergillusniger AAM08362 225 24.1 5.23 22.11 59.78 -0.396
21 Aspergillusniger XP_001389848 231 24.8 3.94 26.61 61.69 -0.364
22 Aspergillusniger GAQ35804 231 24.8 3.93 25.84 61.26 -0.373
23 Aspergillusniger GAQ46944 211 22.5 4.07 17.72 60.52 -0.138
24 Aspergillusniger EHA24718 236 25.7 4.4 21.78 65.64 -0.408
25 Aspergillusniger ALN49265 256 28 4.7 34.64 70.04 -0.312
26 Aspergillusniger AFK10490 211 22.5 4.31 23.47 58.72 -0.146
27 Aspergillusniger ADO66655 211 22.6 4.31 22.55 58.72 -0.145
28 Aspergillusniger000000 XP_001401361 211 22.6 4.31 23.06 58.72 -0.156
29 Aspergillusniger ACN82438 211 22.5 4.39 23.33 57.35 -0.17
30 Aspergillusniger GAQ40597 256 28 4.76 34.57 70.78 -0.303
31 Aspergillusoryzae XP_001823798 232 24.4 5.48 21.33 59.52 -0.276
32 Aspergillusoryzae XP_001818666 221 23.7 4.67 19.63 61.76 -0.424
33 Aspergillusclavatus XP_001273882 192 20.4 9.63 32.86 57.92 -0.357
34 Aspergillusluchuensis GAT31123 225 24.1 5.74 22.92 60.22 -0.432
35 Aspergillusluchuensis GAT25039 211 22.6 4.07 20.58 59.15 -0.129
36 Aspergillusluchuensis OJZ80582 211 22.6 4.02 21.52 57.77 -0.153
37 Aspergillusluchuensis GAT30893 256 28 4.76 34.57 69.26 -0.304
38 Aspergillusluchuensis OJZ85846 256 28 4.84 35.16 68.87 -0.308
39 Aspergillusversicolor ABM55503 206 21.9 4.24 16.32 62.96 -0.065
40 Fusariumavenaceum KIL90649 287 29.8 4.53 29.56 42.93 -0.625
41 Fusariumverticillioides XP_018753195 314 32.6 4.64 24.61 37.96 -0.832
42 Fusariumverticillioides XP_018753196 296 30.6 4.87 21.85 39.93 -0.749
43 Fusariumverticillioides XP_018755440 233 25.2 8.98 30.08 58.93 -0.436
44 Fusariumverticillioides XP_018761356 231 25.7 6.41 34.6 51.95 -0.694
45 Fusariummangiferae CVK98696.1 231 25.6 6.41 34.56 51.52 -0.685
46 Fusariumproliferatum CVL11933 231 25.6 6.05 36.73 53.2 -0.646
47 Fusariumfujikuroi CCT69575 231 25.7 6.41 36.04 53.2 -0.662
48 Fusariumpseudograminearum XP_009253878 231 25.7 6.5 32.22 52.79 -0.666
49 Fusariumlangsethiae KPA38457.1 228 24.5 9.17 26.82 58.64 -0.472
50 Fusariumoxysporum EWZ00952 277 29.2 4.91 23.97 41.59 -0.736
51 Fusariumoxysporum EWZ46984 289 30.3 4.83 24.9 41.87 -0.754
52 Fusariumoxysporum f. sp.vasinfectum EXM22239 271 28.3 5.19 22.81 43.21 -0.676
53 Fusariumoxysporum f. sp.vasinfectum EXM22238 289 30.3 4.83 25.75 40.87 -0.77
54 Fusariumoxysporum f. sp. lycopersici XP_018238679 277 28.8 5.06 23.67 42.64 -0.686
55 Fusariumoxysporum f. sp. lycopersici XP_018238678 286 29.7 4.98 24.79 40.63 -0.737
56 Fusariumoxysporum f. sp. lycopersici XP_018246999 232 25.1 8.96 25.04 57.46 -0.479
57 Fusariumoxysporum f. sp. lycopersici XP_018256151 231 25.6 6.18 32.52 52.77 -0.655
58 Fusariumoxysporum f. sp. cubense EMT73821 231 25.6 6.41 34.17 54.46 -0.638
BACTERIA
59 Bacillus cereus AAZ17391 213 23.3 9.44 15.87 54.46 -0.425
60 Dictyoglomusthermophilum WP_012547705 360 39.7 8.68 25.12 72.28 -0.314
61 Dictyoglomusthermophilum AAC46361 360 39.7 8.68 19.75 71.19 -0.333
62 Dictyoglomusturgidum WP_012582654 356 39.4 8.45 22.94 71.99 -0.285
63 Fibrobactersuccinogenes WP_014546846 327 36.1 5.07 19.20 65.32 -0.350
64 Paenibacillusjilunlii WP_062524300 212 23.2 9.10 24.53 56.08 -0.342
65 Paenibacilluspolymyxa ADK47978 211 22.7 9.55 17.82 61.47 -0.303
66 Paenibacilluspolymyxa KOS03251 212 23.2 9.40 21.41 51.93 -0.407
67 Paenibacilluspolymyxa WP_025720875 212 23.1 9.40 21.41 51.93 -0.419
68 Paenibacilluspolymyxa WP_061831741 212 23.1 9.15 21.05 51.93 -0.417
69 Paenibacilluspolymyxa WP_013308993 212 23.2 9.15 20.83 51.93 -0.432
70 Paenibacilluspolymyxa WP_016820426 212 23.1 9.30 22.35 55.14 -0.409
71 Paenibacilluspolymyxa WP_017425612 212 23.2 9.30 22.05 53.30 -0.417
72 Paenibacilluspolymyxa WP_023987219 212 23.1 8.94 20.56 51.93 -0.414
73 Paenibacilluspolymyxa WP_031462284 212 23.1 9.30 21.95 55.14 -0.407
74 Paenibacilluspolymyxa WP_013373220 212 23.2 9.40 21.91 53.77 -0.407
75 Paenibacilluspolymyxa WP_058831015 212 23.1 9.40 19.84 55.61 -0.394
76 Paenibacilluspolymyxa WP_039272535 212 23.1 9.18 23.35 52.41 -0.404
77 Paenibacilluspolymyxa WP_064797296 212 23.1 9.40 16.99 53.77 -0.445
78 Paenibacilluspolymyxa WP_023987332 362 39.5 7.69 26.27 59.78 -0.524
79 Paenibacilluspolymyxa WP_068938485 362 39.5 7.69 25.82 59.50 -0.525
80 Paenibacilluspolymyxa WP_071639791 362 39.5 7.69 19.53 62.18 -0.504
81 Paenibacillusriograndensis WP_020430448 212 23.1 9.25 20.64 56.56 -0.316
82 Paenibacillusriograndensis WP_060864761 212 23.2 9.25 20.73 56.08 -0.320
83 Paenibacillus terrae WP_014280040 212 23.1 9.30 21.81 52.41 -0.405
ACTINOMYCETES
84 Actinobacteria WP_054228265 330 35.0 9.17 28.72 51.42 -0.415
85 Hamadaeatsunoensis WP_027341164 327 33.8 9.16 26.33 56.70 -0.309
86 Herbidosporacretacea WP_061296570 321 34.0 9.49 29.13 50.16 -0.513
87 Herbidosporamongoliensis WP_066360436 322 33.8 9.37 25.86 48.79 -0.493
88 Microbispora sp. WP_055478269 335 35.5 9.69 32.99 49.55 -0.569
89 Micromonosporacoxensis SCG34253 330 34.5 9.42 33.72 53.82 -0.366
90 Micromonosporanigra SCL32394 329 34.5 9.61 32.39 54.83 -0.395
91 Nocardiopsisdassonvillei WP_061080181 332 35.2 8.74 35.03 51.99 -0.497
92 Nonomuraeajiangxiensis SDH13383 321 33.9 9.32 36.29 56.20 -0.397
93 Planomonosporasphaerica WP_068897915 335 35.1 9.60 32.76 48.39 -0.503
94 Saccharothrixsyringae WP_033431747 329 34.8 9.67 30.96 53.98 -0.495
95 Streptomonospora alba WP_040275156 339 35.8 5.17 35.81 44.31 -0.605
96 Streptomyces hirsutus WP_055594006 337 35.9 9.30 26.42 52.37 -0.406
97 Streptomyces reticuli WP_059255807 337 35.8 9.53 29.68 54.69 -0.413
98 Streptomyces viridosporus AAF09501 329 35.1 9.55 26.02 50.09 -0.518
99 Streptomyces davawensis WP_015659056 320 33.8 9.23 23.06 56.38 -0.351
100 Streptomyces aureus WP_051901343 313 32.6 8.95 20.17 52.36 -0.394
101 Thermobifidafusca WP_011291660 338 36.4 9.47 34.31 52.28 -0.495
102 Thermobifidafusca WP_016188539 338 36.4 9.37 34.20 52.28 -0.504
YEAST
103 Aureobasidiummelanogenum KEQ63689 218 23.3 4.73 30.22 70.78 -0.204
104 Aureobasidiummelanogenum KEQ63789 217 23.4 8.27 20.93 52.63 -0.369
105 Aureobasidiummelanogenum KEQ64351 221 23.4 4.86 17.74 60.90 -0.108
106 Aureobasidiummelanogenum BAB69655 221 23.3 4.86 17.74 61.36 -0.096
107 Aureobasidiumnamibiae XP_013425857 225 24.1 9.25 24.83 60.71 -0.432
108 Aureobasidiumnamibiae XP_013422490 218 23.1 6.40 25.16 72.11 -0.204
109 Aureobasidiumnamibiae XP_013429521 221 23.2 5.71 18.17 62.26 -0.138
110 Aureobasidiumpullulans KEQ83780 229 25.0 8.84 22.97 48.52 -0.514
111 Aureobasidiumpullulans KEQ90048 224 24.1 9.03 24.80 58.39 -0.376
112 Aureobasidiumpullulans KEQ80629 218 23.2 5.54 35.83 75.23 -0.176
113 Aureobasidiumpullulans AAD51950 221 23.5 5.29 16.84 58.73 -0.181
114 Aureobasidiumsubglaciale XP_013347844 224 24.1 8.84 29.17 58.35 -0.420
115 Aureobasidiumsubglaciale XP_013339677 230 25.0 7.77 34.07 52.57 -0.464
116 Baudoiniapanamericana XP_007672582 188 20.3 6.54 23.04 61.22 -0.298
117 Bispora sp. ADZ99365 205 21.8 4.21 14.67 59.41 -0.281
118 Cryptococcus sp. BAA09699 209 22.7 5.46 19.73 50.81 -0.515
119 Cryptococcus sp. BAA09698 209 22.7 5.46 19.73 50.81 -0.515
120 Pseudozymahubeiensis XP_012187186 261 27.9 9.15 24.47 50.84 -0.466
121 Saitozymaflava AOS95422 209 22.7 6.25 18.09 51.29 -0.499
122 Saitozymaflava ABY50453 209 22.7 6.25 21.81 52.20 -0.506

The stability –instability index method26(Guruprasad et al. 1990) estimates the stability of the protein in a test tube. The instability index less than 40 predicts a stable protein whereas values higher than 40 denotes potentially unstable protein. The value of instability index of xylanases ranged from 14.67-36.73, which is less than 40 and hence represents stable protein.The stability -aliphatic index method20(Gasteiger et al., 2005) reflects regional stability based on the relative volume occupied by aliphatic side chain and is a positive indicator of globular protein theromostability.The aliphatic index of xylanaseis the range of 37.96-75.23 as reported in literature27(Walia et al., 2015) and high aliphatic index indicates stability of xylanases for wide temperature range. The aliphatic index of xylanases protein sequences from Aspergillusniger (ALN49265), Dictyoglomusthermophilum(WP_012582654, AAC46361), Aureobasidiummelanogenum (KEQ63689), Aureobasidiumnamibiae (XP_013422490) and Aureobasidiumpullulans(KEQ80629) was above 70 (Table-1).Another important physio-chemical attributeanalyzed by ProtParam is GRAVY value derived by calculating the sum of hydropathy values28(Kyte and Doolittle, 1982) of all the amino acids, divided by the number of residues in the sequence20(Gasteiger et al. 2005). Increasing positive score indicates a greater hydrophobicity. The microbial xylanase protein sequences revealed negative GRAVY value ranging from -0.832 to -0.093 indicating hydrophilic nature.

Multiple Sequence Alignment Analysis

Multiple sequence alignment ofretrievedxylanasesequenceswasperformed by CLUSTAL X version 2.1andis shown in Figure 1(A,B,C& D). Several conserved amino residues are observed for different source organisms while comprehensive multiple sequence alignment of all122xylanase sequences revealedtwohighlyconserved residues namely YGW and EYYI (Figure-1E). The presence of these conserved amino acid residues has been reported for xylanases especially from fungal and bacterial sources29-31(Ellouze et al.,2011, Sapag et al., 2002,Torronen et al.,1992).Similar conserved amino acid residues have been observed for xylanaseofT. longibrachiatum. Another conserved amino acid residues with sequence RVNEPSIQGTATFNQYhas been reported, which plays significant role in stabilizing during substrate binding16(Uzuner et al.,2010).

Figure 1: Multiple sequence alignment of xylanase protein sequences from (A) Fungal (B) Bacterial (C) Actinomycetes and (D) Yeast sources.Strongly conserved amino acid residues are indicated by asterisk* above the alignment.

Figure 1: Multiple sequence alignment of xylanase protein sequences from (A) Fungal (B) Bacterial (C) Actinomycetes and (D) Yeast sources.Strongly conserved amino acid residues are indicated by asterisk* above the alignment.

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 Figure-1.e: Combined sequence alignment of a total 122xylanases protein sequences from different microbial sources.Strongly conserved amino acid residues are indicated by asterisk* above the alignment.

Figure 1.e: Combined sequence alignment of a total 122xylanases protein sequences from different microbial sources.Strongly conserved amino acid residues are indicated by asterisk* above the alignment.

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It has also been reported that glutamate amino acid residue responsible for catalysis is conserved in genera of ascomycetes and basidiomycetes representing GH11 and GH10 family of xylanases32(Cervanteset al., 2016).Xylanase proteins representing actinomycetes revealed several conserved amino acid residues at positions 119-131,155-169,185-191,197-208 and 221-233 along with YGW and EYY residues (Figure-1C). Similarly alignment of 20 xylanase protein sequences of GH11 family from source organism yeast revealed several conserved amino acid residues at position 214-216 (Figure-1D). The presence of conserved amino acid residues provides an insight into the catalytic activity of the enzyme based on the fact that there exits sequence-structure-function relationship. The multiple sequence alignment also provides an opportunity to design appropriate degenerate primers for amplification of xylanase genes from different microbial sources.

Phylogenetic Analysis

The phylogenetic tree based on microbial xylanase protein sequenceswere constructed by NJ method (Figure-2 A, B, C, D). The phylogenetic tree representing fungal xylanase protein sequences revealed 5 distinct major clusters designated as I,II,III,IV and V group (Figure-2A).

 Figure 2.a: Phylogenetic tree constructed using protein sequences of 58 fungal xylanase. The distinct major clusters designated as I, II, III, IV and V comprising of19, 19, 5, 10 and 5 members respectively are highlighted

Figure 2.a: Phylogenetic tree constructed using protein sequences of 58 fungal xylanase.

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The distinct major clusters designated as I, II, III, IV and V comprising of19, 19, 5, 10 and 5 members respectively are highlighted

Genera specific clusters for different species of Aspergillus and Fusarium were observed. This indicates sequence level similarity among xylanases representing specific genera and could be utilized to decipher specific sequence features for designing genera specific probe or primers exclusively for xylanase genes. Further distinct sub-clusters representing multiple strains of predominately Aspergillusniger and Fusariumoxysporum were also observed (Figure-2A). In case of bacterial xylanases two distinct clusters designated as I and II comprising exclusively forPaenibacillusandDictyoglomus species were observed (Figure-2B).

 Figure 2.b: Phylogenetic tree constructed using 25 protein sequences of xylanasesfrom bacterial sources. The distinct major clusters designated as I and II comprising of 21 and 3 members respectively are highlighted.

Figure 2.b: Phylogenetic tree constructed using 25 protein sequences of xylanasesfrom bacterial sources.

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The distinct major clusters designated as I and II comprising of 21 and 3 members respectively are highlighted.

Xylanase from Fibrobactersuccinogenes occupied distinct place in the phylogenetic tree. The major clusters I and II represented predominantly multiple strains of Paenibacilluspolymyxa and Dictyoglomusthermophilum indicating strain specific sequence similarity.Similarly, the phylogenetic tree for xylanasesfromactinomycetes revealed two major clusters I and II with 15 and 4 members respectively. The major cluster I was further divided into three subclusters i.e. A, B, C (Figure-2C).

 Figure 2.c: Phylogenetic tree constructed using 19 xylanaseprotein sequences of actinomycetes.The distinct major clusters designated as I and II comprising of 15 and 4 members respectively are highlighted.

Figure 2.c: Phylogenetic tree constructed using 19 xylanaseprotein sequences of actinomycetes.

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The distinct major clusters designated as I and II comprising of 15 and 4 members respectively are highlighted.

In case of xylanases from yeast sources, two major clusters I and II with 12 and 8 sequences were observed, which were further divided into two sub-clusters A and B respectively (Figure-2D).The phylogenetic tree comprising of all the 121 sequences representing different microbial sources revealed seven distinct major clusters designated as A, B, C, D, E, F and G. These major clusters represented specific source organisms (Figure-2D). The major cluster A comprising of 19 sequences represents actinomycetes source organism exclusively while B represented bacterial sources. The major cluster C with 12 sequences represents both bacterial and fungal sources while D comprises of 22 sequences exclusively from Aspergillus genera.

 Figure 2.d: Phylogenetic tree constructed using 20 xylanase protein sequences of yeast.The distinct major clusters designated as I and II comprising of 12 and 8 members respectively are highlighted.

Figure 2.d: Phylogenetic tree constructed using 20 xylanase protein sequences of yeast.

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The distinct major clusters designated as I and II comprising of 12 and 8 members respectively are highlighted.

The major cluster E included 3 sequences of yeast genera, F included 21 sequences predominantly from Fusariumgenera along with some sequences from yeast and the major cluster G with 27 sequences comprises of both fungal and yeast source organisms (Figure-2D).  Phylogenetic tree revealing xylanases representing GH10 and GH11 family and also basidiomycetes and ascomycetes specific fungal groups have been reported32,29(Cervanteset al.,2016; Ellouzeet al.,2011). Distinct clades representing GH10, GH11 and GH30 family revealing evolutionary relatedness based on 22 protein sequences of xylanaseswerealso deciphered33(Liao et al., 2015).

Figure 2.e: Phylogenetic tree constructed using 122 protein sequences of xylanase representing different microbial sources. The major clusters designated as A,B,C,D,E,F and G is highlighted.

Figure 2.e: Phylogenetic tree constructed using 122 protein sequences of xylanase representing different microbial sources.

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The major clusters designated as A,B,C,D,E,F and G is highlighted.

Motif distribution And characterization

The conserved motifs deduced by MEME are generally analyzed for biological function using protein BLAST and domains are characterized by Interproscanto reveal the best possible match based on highest similarity score.The distribution of five motifs among microbial xylanase protein sequencesis shown in Figure 3A, B, C and D.

 Figure 3: Distribution of 5 commonly observed motifs among xylanases representing different microbial sources(A) Fungal (B) Bacterial (C) Actinomycetes and (D) Yeast sources.

Figure 3: Distribution of 5 commonly observed motifs among xylanases representing different microbial sources (A) Fungal (B) Bacterial (C) Actinomycetes and (D) Yeast sources.

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The distribution of five motifs among 58 fungal xylanase protein sequences was analyzed (Figure3A) and motifs with width and best possible match amino acid sequences is shown in Table-2A. The predominance of motifs with conserved domain representing unique feature of GH11 family was observed. The motif 1 with amino acid sequence IDGTATFTQYWSVRQNKRSSGTVTTSNHFNAWAKLGMNLGTHNYQIVATE and motif 2 with sequence PSGNGYLSVYGWTTNPLVEYYIVESYGTYNPGSGGTYKGTV was uniformly distributed among fungal xylanases. Similarly the motif assessment for bacterial(Figure-3B, Table-2B), actinomycetes (Figure-3C, Table-2C) and yeast (Figure-3D, Table-2D) source organisms revealed predominance of conserved domains specific to GH11 family.

Table 2.a: The best possible match amino acid sequences of five motif with respective domains observed for xylanasesinFungal, Bacterial, Actinomycetes and Yeast.

Motif no. Sequencelength Sequence Occurrence at different site Conserved

Domain

    (A) FUNGAL    
1 50 IDGTATFTQYWSVRQNKRSSGTVTTSNHFNAWAKLGMNLGTHNYQIVATE 58      GH11family
2 41 PSGNGYLSVYGWTTNPLVEYYIVESYGTYNPGSGGTYKGTV 58       GH11family
3 21 GNFVGGKGWNPGSARTITYSG 56       GH11family
4 21 NNGFYYSFWTDGGGDVTYTNG 47       GH11family
5 15 DGSTYDIYTTTRTNA 57      No information
    (B) BACTERIAL    
1 41 AGVWAPSGNGYLALYGWTRNSLIEYYVVDSWGTYRPTGTYK 25 GH11family
2 21 SDGGTYDIYTTMRYBAPSIEG 24 GH11family
3 29 ITFSNHVKAWASKGMNLGSNWSYQVLATE 21 GH11family
4 21 AATDYWQNWTDGGGTVNAVNG 21 No information
5 29 GGNYSVTWKBTGNFVVGKGWTTGSPNRTI 21 GH11family
    (C) ACTINOMYCETES    
1 50 YDIYKTTRYNAPSIEGTRTFDQYWSVRQSKRTGGTITSGNHFDAWARAGM 19  GH11family
2 50 RRSVTYSGSFNPSGNAYLTLYGWTRNPLVEYYIVDNWGTYRPTGTYKGTV 19  GH11family
3 50 CTATLSAGQQWSDRYNLNVSVSGSSNWTVTMNVPSPAKVJSTWNVSASYP 19 No information
4 50 VTTNQTGTNNGYFYSFWTDSQGTVSMELGSGGNYSTSWRNTGNFVAGKGW 18  GH11family
5 29 LTARPNGNGNNWGVTIQHNGNWTWPTVSC 19 No information
    (D) YEAST    
1 50 SDGSTYDVCTDTRTNQPSITGTSTFKQYWSVRQNKRTSGTVTTQNHFNYW 20  GH11family
2 16 WTNSPLVEYYVIESYG 20  GH11family
3 23 GSYNYQVMATEGFSGSGSASVTV 19  GH11family
4 21 NTDFVVGLGWSTGAARTITYS 20 GH11family
5 29 INYVQNYNGNVANFTYNZNAGTYSMNWNN 12 No information

The comprehensive analysis of all the xylanases sequences, irrespective of source organisms for motif distribution and domain characterization is shown in Figure-3E and Table-2B respectively. A total of 10 motifs among the 122 xylanase protein sequences revealed 6 motifs with domains specific to GH11 family. The Motif 3 with sequence GTVTSDGGTYDIYTTTRTNAP was found to be highly conserved and was found uniformly among most of the microbial xylanase sequences analyzed. The sequence motifs could be considered as signature sequence revealing the functional identity of the proteins or enzymes and could be targeted for enzyme engineering. The motif assessment also provides an insight into the structural and functional diversity of the enzymesas reported34 (Mohammed et al., 2011). Motif assessment for Endo-1,4-β-xylanase of  GH11 family from source organism Paecilomycesvariotii, Schizophyllum commune and Trichodermaharzianumhas been reported15(Arora et al.,2009).

Figure 3.e: Distribution of 10 commonly observed motifs among 122 xylanase protein sequences.

Figure 3.e: Distribution of 10 commonly observed motifs among 122 xylanase protein sequences.

Click here to View figure

Table 2.b: The best possible match amino acid sequences of 10 motifs with respective conserved domain observed among 122 protein sequences of xylanases from different microbial sources.

Motif no. Sequence length Sequence Occurrence at different site Conserved

Domain

1 21 NSYLAVYGWTRNPLVEYYIVE 118 GH 11Family
2 18 IDGTATFTQYWSVRQSKR 118 GH 11Family
3 21 GTVTSDGGTYDIYTTTRTNAP 121 GH11Family
4 17 TVTTGNHFBAWASLGMN 120 GH11Family
5 21 HBYQILATEGYQSSGSSSITV 120 GH11Family
6 15 WSNTGNFVGGKGWNT 115  No information
7 29        NNGYYYSFWTDGGGTVTYTNGSGGNYSVE 84 GH11Family
8 50   CTATLSAGQQWSDRYNLNVSVSGSSNWTVTMNVPSPAKVJSTWNVSASYP 19 No information
9 15 SARTITYSGSFNPSG 120 No information
10 11 SYGTYNPGSGY 119 No information

The relevance of bioinformaticsin enzyme engineering has been witnessed in recent years and several in-silico tools mainly focusing on prediction of three dimensional structure of enzyme based on the availability of the protein sequences is now being routinely used35,36(Damborsky and Brezovsky, 2014; Suplatov et al., 2015). The in-silico analysis of the sequences of genes/proteins of several industrially important enzymes mainly focusing on homology search, multiple sequence alignment, phylogenetic tree construction and motif assessment has been reported.37-48(Yadavet al., 2009; Dubeyet al., 2010; Yadavet al., 2010; Malviyaet al., 2011; Dubey et al., 2012; Moryaet al., 2012; Yadav et al., 2012; Kumar et al., 2012; Dwivedi and Mishra, 2014; Mathew et al., 2014; Moryaet al., 2016;; Yadavet al., 2017).

Molecular cloning of relevant genes coding for enzymes and its expression needs bioinformatics interventiontargeting forsubstantial improvement in enzyme for desired features.Recently,functional diversity of multiple xylanases from Penicilliumoxalicum GZ-2, revealing functional redundancy using bioinformatics approach has been reported.33(Liao et al., 2015)

Conclusions

Using bioinformatics approach, an attempt has been made to characterize microbial xylanase sequences for several important attributes, which could be targeted for enzyme engineering to develop novel xylanases. The knowledge about the sequences is being applied for deciphering the three dimensional structure using appropriate in-silicotoolsprior to wet-lab experimentation. The tools of bioinformatics are also relevant in the era of genomics, where several microbial genome sequences have been deciphered. This provides an opportunity to perform genome-wide identification and characterization of multigene families of industrially important enzymes and analyze the functional redundancy.There has been substantial improvement in advanced enzyme technologies including metagenomics and directed evolution based on recent bioinformatics driven approaches.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgments

DA would like to acknowledge the UGC Rajiv Gandhi National fellowship, New Delhi. The authors wish to acknowledge the Head, Department of Biotechnology, D.D.U. Gorakhpur University, Gorakhpur for providing the infrastructural support.

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