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 GH11^3-5 (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 years^2,6-10 (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

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 sources^29-31(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. Click here to View figure

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. Click here to View figure

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. Click here to View figure

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. Click here to View figure

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. Click here to View figure

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. Click here to View figure

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 reported^32,29(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. Click here to View figure

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. Click here to View figure

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.

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. 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.

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 used^35,36(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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