Genetic and functional characterization of a novel GH10 endo-β- 1,4-xylanase with a ricin-type β-trefoil domain-like domain from Luteimicrobium xylanilyticum HY-24

Accepted Manuscript Title: Genetic and functional characterization of a novel GH10 endo-␤-1,4-xylanase with a ricin-type ␤-trefoil domain-like domain from Luteimicrobium xylanilyticum HY-24 Authors: Do Young Kim, Sun Hwa Lee, Min Ji Lee, Han-Young Cho, Jong Suk Lee, Young Ha Rhee, Dong-Ha Shin, Kwang-Hee Son, Ho-Yong Park PII: DOI: Reference:

S0141-8130(17)31736-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.08.063 BIOMAC 8055

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

15-5-2017 9-8-2017 9-8-2017

Please cite this article as: Do Young Kim, Sun Hwa Lee, Min Ji Lee, Han-Young Cho, Jong Suk Lee, Young Ha Rhee, Dong-Ha Shin, KwangHee Son, Ho-Yong Park, Genetic and functional characterization of a novel GH10 endo-␤-1,4-xylanase with a ricin-type ␤-trefoil domain-like domain from Luteimicrobium xylanilyticum HY-24, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.063 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Genetic and functional characterization of a novel GH10 endo-β-1,4-xylanase with a ricin-type β-trefoil domain-like domain from Luteimicrobium xylanilyticum HY-24

Do Young Kima, Sun Hwa Leea, Min Ji Leea, Han-Young Choa, Jong Suk Leeb, Young Ha Rheec, Dong-Ha Shind, Kwang-Hee Sona,*, Ho-Yong Parka,*

a

Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB),

Daejeon 34141, Republic of Korea b

c

Gyeonggi Bio-Center, Gyeonggi Institute of Science & Technology Promotion, Suwon 16229, Republic of Korea

Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, Republic of

Korea d

Insect Biotech Co. Ltd., Daejeon 34054, Republic of Korea

*

Corresponding authors at: Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and

Biotechnology (KRIBB), Daejeon 34141, Republic of Korea. Tel.: +82 42 8604650; fax: +82 42 8604659. E-mail adresses: [email protected] (K.-H. Son); [email protected] (H.-Y. Park).

ABSTACT The gene (1488-bp) encoding a novel GH10 endo--1,4-xylanase (XylM) consisting of an N-terminal catalytic GH10 domain and a C-terminal ricin-type -trefoil lectin domain-like (RICIN) domain was identified from Luteimicrobium xylanilyticum HY-24. The GH10 domain of XylM was 72% identical to that of Micromonospora lupini endo--1,4-xylanase and the RICIN domain was 67% identical to that of Actinospica robiniae hypothetical protein. The recombinant enzyme (rXylM: 49 kDa) exhibited maximum activity toward beechwood xylan at 65 oC and pH 6.0, while the optimum temperature and pH of its C-terminal truncated mutant (rXylM△RICIN: 35 kDa) 1

were 45 oC and 5.0, respectively. After pre-incubation of 1 h at 60 oC, rXylM retained over 80% of its initial activity, but the thermostability of rXylM△RICIN was sharply decreased at temperatures exceeding 40 oC. The specific activity (254.1 U mg-1) of rXylM toward oat spelts xylan was 3.4-fold higher than that (74.8 U mg-1) of rXylM△RICIN when the same substrate was used. rXylM displayed superior binding capacities to lignin and insoluble polysaccharides compared to rXylM△RICIN. Enzymatic hydrolysis of -1,4-D-xylooligosaccharides (X3-X6) and birchwood xylan yielded X3 as the major product. The results suggest that the RICIN domain in XylM might play an important role in substrate-binding and biocatalysis.

Keywords: Luteimicrobium xylanilyticum HY-24 GH10 endo-β-1,4-xylanase Ricin-type -trefoil lectin domain-like domain Substrate-binding Biocatalysis

1. Introduction

Like cellulosic biomass-degrading microorganisms [1], a variety of bacterial and fungal species capable of decomposing hemicelluloses, such as -1,4-D-xylan, -1,4-D-mannan, and -1,5-L-arabinan, are widely distributed in nature [2]. They generally produce diverse exo- and endo-type glycoside hydrolases (GHs) to completely cleave glycosidic bonds present in the backbone of polysaccharide chains by a concerted action. Of such hemicelluloses in hardwood trees, -1,4-D-xylan is a major structural polysaccharide, in which D-xylose molecules are combined by -1,4-D-xylosidic linkages [3]. Endo-β-1,4-xylanases are primary hydrolytic enzymes responsible for the breakdown of -1,4-D-xylan polysaccharides [2]. These biocatalysts are currently classified into six GH families (5, 8, 10, 11, 30, and 43) based 2

on their primary structural similarities (http://www.cazy.org/Glycoside-Hydrolases.html). In particular, various microbial endo-β-1,4-xylanases with genetic diversity belonging to GH families 10 and 11 have attracted a great deal of industrial attention [3,4]. Compared to GH11 endo-β-1,4-xylanases consisting of a -jelly roll as a catalytic domain, GH10 endo-β-1,4-xylanases share a (/)8-barrel as a catalytic domain [5]. In addition, GH10 enzymes are frequently identified as multi-domain proteins composed of two or more functional domains (such as carbohydratebinding module, fibronectin type 3 domain, and ricin-type -trefoil lectin domain) and linker regions [6]. During the past decade, various lignocellulose-degrading bacteria have been isolated from digestive tracts of the earthworm Eisenia fetida and wood-feeding insects and some of their related enzymes with distinct molecular features have been genetically and functionally characterized [6-11]. Metagenomic sequence analyses have also revealed that gut microbiomes associated with wood-feeding insects, such as beetle and higher termite, contain a particular plant biomass-degrading system [12,13]. Thus, to obtain a novel endo-β-1,4-xylanase with peculiar biochemical and molecular characteristics, we screened and isolated some aerobic -1,4-D-xylan-decomposing microorganisms resident in the digestive tract of the long-horned beetle Massicus raddei. Here, we report the genetic and biocatalytic characteristics of a modular GH10 endo--1,4-xylanase (XylM) with a ricin-type -trefoil lectin domain-like (RICIN) domain from Luteimicrobium xylanilyticum HY-24 (=W-15T =KCTC 19882T) [14], a symbiotic bacterium in the gut of M. raddei. The alterations of enzymatic properties of the recombinant enzyme (rXylM) induced by deleting a C-terminal RICIN domain and the functional role of the RICIN domain with respect to -1,4-D-xylan degradation are also described.

2. Materials and methods

2.1. Chemicals

A series of β-1,4-D-xylooligosaccharides (X2-X6) and ivory nut mannan were obtained from Megazyme International Ireland Ltd. (Wicklow, Ireland). Chitosan was provided by USB Co. (Cleveland, OH, USA). All other 3

substances including sugars (D-cellobiose, D-glucose, D-mannose, and D-xylose) substituted with p-nitrophenol (PNP), D-xylose (X1), and β-1,4-D-xylans from beechwood, birchwood, and oat spelts were purchased from SigmaAldrich (St. Louis, MO, USA).

2.2. Cloning of the endo--1,4-xylanase (XylM) gene

To amplify a partial sequence of the XylM gene from the genomic DNA of L. xylanilyticum HY-24, polymerase chain reaction (PCR) was conducted using degenerate oligonucleotides designed based on conserved regions (WDVVNE and ITELDV) in the GH10 endo--1,4-xylanases. The upstream oligonucleotide (XylM-F) was 5’TGGGACGTCSTCAACGAG-3’

and

the

downstream

oligonucleotide

(XylM-R)

was

5’-

GACGTCGAGCTCSGTGAT-3’, which yielded a 336-bp DNA fragment. The complete gene encoding XylM was obtained by repeated genomic walking and nested PCR methods using a DNA Walking SpeedUpTM Premix Kit (Seegene, Seoul, Republic of Korea). It was then cloned into a pGEM®-T Easy vector (Promega, Madison, WI, USA).

2.3. Expression and purification of rXylM and rXylM△RICIN

To overproduce a mature recombinant protein (rXylM), its encoding gene with NdeI restriction site in the Nterminal region and HindIII restriction site in the C-terminal region was amplified by PCR using the pGEM®-T Easy/xylM

vector

as

a

template.

The

PCR

primers

used

were

as

follows:

mXylM-F

(5’-

CATATGGCAACGCCCGCACAGG-3’) and mXylM-R (5’-AAGCTTTCAGTGGCTGGTCAGGGTCC-3’). The PCR reaction was conducted using a DNA thermal cycler (TaKaRa) with a 50 L PCR mixture [20 ng of template DNA, 100 pmol of each primer, 2.5 mM of each dNTP, 2.5 units of FastStart Tag DNA polymerase (Roche), a GCrich solution, and a PCR buffer]. The initial template denaturation was done for 4 min at 95 oC. This was followed by 35 cycles of 30 sec at 95 oC, 30 sec at 54 oC, and 1 min 30 sec at 72 oC. Electrophoretic separation of the 4

amplified gene products was performed on a 1.2% agarose gel and the corresponding gene products were then purified using a NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany). The obtained PCR products were cloned into a pGEM®-T Easy vector, followed by excising the recombinant vectors with NdeI and HindIII to produce the xylM fragments with the corresponding sticky ends. The generated gene products were isolated and ligated into a pET-28a(+) vector (Novagen) with the same ends, after which transformation of the resulting pET-28a(+)/xylM into Escherichia coli BL21 was accomplished. Similarly, PCR amplification of the gene encoding

XylM△RICIN

was

performed

CATATGGCAACGCCCGCACAGG-3’)

using

the

and

following

primers

mXylM△RICIN-F

mXylM△RICIN-R

(5’(5’-

AAGCTTTCAGTTGATCGTGCTCCCACCG-3’), according to the method with minor modifications as described above. In this case, the PCR reaction was carried out for 35 cycles of 30 sec at 95 oC, 30 sec at 54 oC, and 1 min at 72 oC. Construction of recombinant E. coli BL21 harboring pET-28a(+)/xylM△RICIN was accomplished as demonstrated above. Overproduction of rXylM and rXylM△RICIN was performed by culturing the recombinant E. coli BL21 cells containing pET-28a(+)/xylM or pET-28a(+)/xylM△RICIN using a 2-L baffle-flask, which contained 0.5 L of Luria-Bertani broth (Difco) and 25 mg/L of kanamycin, in a rotary shaker (150 rpm) for 12 h at 30 oC. The expression of the XylM and XylM△RICIN genes was induced by adding 1 mM IPTG after the absorbance of the culture at 600 nm reached approximately 0.45. After cultivation, the XylM- or XylM△RICIN-expressing cells were collected by centrifugation (5,000 x g) and suspended in binding buffer (20 mM imidazole, 0.5 M NaCl, and 20 mM sodium phosphate) (pH 7.4). Disruption of the recombinant cells was conducted by sonication. The soluble fraction containing active rXylM or rXylM△RICIN proteins was recovered by centrifugation (12,000 x g) for 15 min at 4 oC, filtered, and then directly used as the crude enzyme preparation. Purification of the recombinant enzymes was subjected to affinity column chromatography using a HisTrap HP (GE Healthcare, Uppsala, Sweden) (5 mL) column attached to a fast-protein liquid chromatography system (Amersham Pharmacia Biotech, Uppsala, Sweden), according to the manufacturer’s instructions. The N-terminal (His)6-tagged proteins were eluted from the column employing a linear gradient of 20–500 mM imidazole at a flow rate of 2 mL min-1. The active fractions were then recovered and desalted with a HiPrep 26/10 desalting column (Amersham Biosciences) using 50 mM 5

sodium phosphate buffer (pH 6.0) as the mobile phase. The fractions with high endo--1,4-xylanase activity were collected and subjected to further analysis. The relative molecular mass of purified rXylM and rXylM△RICIN was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the denatured proteins using a 12.0% gel. After SDS-PAGE analysis, the gel was stained with Coomassie brilliant blue R-250 to visualize the separated protein bands. Bradford assay was employed to measure the concentration of proteins using bovine serum albumin as a standard [15].

2.4. Enzyme assays

Endo-β-1,4-xylanase activity was assayed by measuring the amount of reducing sugars released from beechwood xylan using 3,5-dinitrosalicylic acid reagent and xylose as a standard [16]. Endo--1,4-xylanase activity of rXylM was assayed using the standard assay mixture (0.5 mL) consisting of 1.0% beechwood xylan and appropriately diluted enzyme solution (0.05 mL) in 50 mM sodium phosphate buffer (pH 6.0) at 65 oC for 15 min. Conversely, the assay of rXylM△RICIN activity was conducted using the standard assay mixture (0.5 mL) consisting of 1.0% beechwood xylan and appropriately diluted enzyme solution (0.05 mL) in 50 mM sodium citrate buffer (pH 5.0) at 45 oC for 15 min. One unit (U) of endo-β-1,4-xylanase activity for β-1,4-D-xylan polysaccharides was defined as the amount of enzyme required to produce 1 µmol of reducing sugar per min under standard assay conditions.

2.5. Effects of pH, temperature, and chemicals on the activity of endo-β-1,4-xylanase

The effect of pH on the endo-β-1,4-xylanase activity of purified rXylM was evaluated by subjecting samples to pHs ranging from 3.5 to 11.0 at 65 oC for 15 min using the following buffer systems (50 mM): sodium citrate (pH 3.5-5.5), sodium phosphate (pH 5.5-7.5), Tris-HCl (pH 7.5-8.5), and glycine-NaOH (pH 8.5-11.0). However, the effect of pH on the activity of purified rXylM△RICIN was determined at 45 oC due to its thermal instability at 65 6

o

C. The effect of temperature on the biocatalytic activities of rXylM and rXylM△RICIN was evaluated at 30, 35,

40, 45, 50, 55, 60, 65, 70, and 75 oC under their respective standard assay conditions. The thermal stabilities of rXylM and rXylM△RICIN were determined by measuring their residual endo-β-1,4-xylanase activities after preincubation of the recombinant enzymes at a range of 30 to 65 oC for 1 h. The effects of metal ions (each 1 mM) and reactive chemicals (each 5 mM) on the endo-β-1,4-xylanase activity of rXylM were assessed after pre-incubation of the enzyme at 4 oC for 10 min in 50 mM sodium phosphate buffer (pH 6.0) containing the compound of interest.

2.6. Identification of the degradation products

Enzymatic degradation of birchwood xylan (10 mg) and -1,4-D-xylooligomers (X2-X6, each 1 mg) was carried out using the purified rXylM (3 μg) in 100 μL of 50 mM sodium phosphate buffer (pH 6.0) for 6 h at 30 oC, during which time the enzyme maintained over 95% of its original biocatalytic activity. The enzyme reaction was then finished by heating the reaction mixtures at 100 oC for 5 min. The degradation products were identified and quantitatively analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS), as previously described [8,17].

2.7. Binding assay

Binding abilities of rXylM and rXylM△RICIN to hydrophobic substrates were assessed using the diverse insoluble polymers with a specific microstructure such as lignin, chitosan, chitin, oat spelts xylan, Avicel PH-101, and ivory nut mannan. Briefly, prior to the binding assay, the hydrophobic polymers were washed five times in 50 mM sodium phosphate buffer (pH 6.0) to eliminate any water-soluble compounds. The binding capacities of two recombinant enzymes to insoluble polymers were then investigated by incubating rXylM or rXylM△RICIN (approximately 5 U mL-1) in 1.5 mL Eppendorf tubes containing an equal volume of hydrophobic substrate on ice for 2 h and stirred every 5 min. After completion of binding experiments, the reaction mixtures were centrifuged, 7

after which the collected supernatant was directly subjected to determination of the remaining enzyme activity and protein concentration.

3. Results and discussion

3.1. Genetic characterization of the novel endo-β-1,4-xylanase gene

The XylM gene (GenBank accession number: KY997451) identified in the present study contained a 1488-bp open reading frame that encodes a polypeptide of 495 amino acids with a calculated pI of 6.49 and a deduced molecular mass of 52,009 Da. As examined using the SignalP 4.1 server, the XylM was predicted to possess a signal peptide in the N-terminus region that might be cleaved between Ala37 to Ala38 (Fig. 1), indicating that this enzyme is an extracellular biocatalyst. Accordingly, the mature XylM was predicted to be an acidic protein having a calculated pI of 5.99 and a deduced molecular mass of 48,186 Da. BLAST and Pfam analyses of the primary structure of XylM revealed that it might be a modular enzyme consisting of two putative functional domains: an Nterminal catalytic GH10 domain (Leu49 to Leu338) and a C-terminal RICIN domain (Leu395 to Trp480) (Fig. 1). A RICIN domain is known as a carbohydrate-binding domain consisting of three homologous subdomains of 40 amino

acids

and

a

linker

sequence

of

around

15

residues

(http://smart.embl.de/smart/do_annotation.pl?DOMAIN=SM00458). The aforementioned domain architecture of XylM was most similar to that of a GH10 endoxylanase (CAD48748) from Thermopolyspora flexuosa that has not yet been biocatalytically characterized to date. In addition, XylM was structurally analogous to some uncharacterized GH10 endo--1,4-xylanases (AAP87538, AAA17888, and AAD32560) identified from the genomes of culturable and unculturable microorganisms. Multiple sequence alignment showed that the catalytic GH10 domain (Leu49 to Leu338) of XylM from L. xylanilyticum HY-24 shared 72%, 72%, 72%, and 69% sequence identities with that of Micromonospora lupini endo--1,4-xylanase

(WP_007459199),

Actinoplanes 8

globisporus

endo--1,4-xylanase

(WP_020516021),

Amycolatopsis mediterranei endo--1,4-xylanase (WP_013225148), and Cellulomonas flavigena endo--1,4xylanase (WP_013115627), respectively (Fig. 1). The maximum sequence identity (67%) of the RICIN domain (Leu395 to Trp480) of XylM was obtained when it was compared to that of Actinospica robiniae hypothetical protein (WP_034263550). Moreover, sequence identities between the RICIN domain of XylM and that of Streptomyces stelliscabiei hypothetical protein (KND40516), Acidobacterium ailaaui hypothetical protein (WP_049961273), and Microbispora rosea RICIN domain (SIR89347) were 64%, 62%, and 59%, respectively. The findings suggest that XylM is a novel endo--1,4-xylanase with peculiar molecular features. The two conserved catalytic residues of Glu172 (acid/base catalyst) and Glu275 (catalytic nucleophile), which may participate in the double-displacement of retaining GH enzymes [18], were found in the active site of premature XylM. The secondary structure elements of XylM from L. xylanilyticum HY-24, which was predicted using a GH10 endo--1,4-xylanase from Streptomyces lividans (PDB code: 1E0V) as a template, are displayed in Fig. 2. The structure-based sequence alignment revealed that the catalytic GH10 domain in XylM was comprised of 12 helices, 3 310-helices, 9 -strands, and 3 -turns. In XylM, the formation of intramolecular disulfide bridges essential for the folding and stability of various biocatalysts was predicted to occur between Cys295 and Cys301 as well as between Cys210 and Cys242.

3.2. Purification and SDS-PAGE analysis of recombinant enzymes

Similar to an extracellular GH10 endo--1,4-xylanase from Cellulosimicrobium sp. HY-13 [6], rXylM and rXylM△RICIN were generally produced as inactive inclusion bodies due to their relatively high hydrophobicity when overexpressed in E. coli BL21. Thus, the two distinct His-tagged proteins with the corresponding biocatalytic activity were purified to electrophoretic homogeneity by an on-column refolding protocol using a His-tag column (Fig. 3). SDS-PAGE analysis showed that the relative molecular mass of purified rXylM was approximately 49 kDa and that of rXylM△RICIN was 35 kDa. These values were in good agreement with the deduced molecular mass (50,480 Da) of rXylM and the deduced molecular mass (35,958 Da) of rXylM△RICIN. The molecular size (49 9

kDa) of rXylM on SDS-PAGE was similar to that (46 kDa) of a modular GH10 endo--1,4-xylanase with a Cterminal cellulose-binding domain (CBD) from Streptomyces sp. S9 [19]. However, rXylM was larger than nonmodular GH10 endo--1,4-xylanases [20,21] with a molecular mass of < 40 kDa, and smaller than a bi-modular GH10 -1,4-xylanase (65 kDa) with a C-terminal -1,4-D-xylan-binding domain from Paenibacillus terrae HPL003 [22].

3.3. Enzymatic properties of rXylM and rXylM△RICIN

Some endo--1,4-xylanases from invertebrate-symbiotic bacteria have been reported to be most specific to 1,4-D-xylans at pHs of 6.0-6.5 [6,9,21]. Similarly, rXylM exhibited maximum biocatalytic activity toward beechwood xylan at 65 oC in 50 mM sodium phosphate buffer (pH 6.0) (Figs. 4a, 5a). Conversely, rXylM△RICIN showed the highest hydrolysis activity toward beechwood xylan at 45 oC in 50 mM sodium citrate buffer (pH 5.0) (Figs. 4b, 5b). It was likely that rXylM was more stable than rXylM△RICIN at pH 6.5-9.5. Especially, the former maintained over 80% of its original biocatalytic activity for beechwood xylan even after pre-incubation for 1 h at a broad pH range of 4.0-8.0. However, the latter retained over 80% of its original biocatalytic activity for the same substrate only between pH 4.0 and 6.0 (Figs. 4c, 4d). rXylM appeared to be relatively thermostable because its residual beechwood xylan-degrading activity exceeded 85% even after pre-incubation for 1 h at 60 oC in the absence of the substrate (Fig. 5). On the other hand, the thermal stability of rXylM△RICIN was gradually reduced in a temperature-dependent manner when it was exposed to a temperature exceeding 40 oC for 1 h. Taken together, these results strongly indicate that the removal of a C-terminal RICIN domain in XylM significantly influenced reduction of its pH stability and thermostability as well as alteration of its optimum pH and temperature for degradation of beechwood xylan. It has also been reported that like the RICIN domain, the deletion of N-terminal carbohydrate-binding modules (CBMs) in an extracellular GH10 endo--1,4-xylanase from Caldicellulosiruptor kronotskyensis negatively affects its optimum temperature and thermostability [23]. The biocatalytic activity of rXylM toward beechwood xylan was slightly upregulated (approximately 1.2-fold) 10

in the presence of a non-ionic surfactant (0.5%), such as Triton X-100 or Tween 80, as has also been shown for some other GH10 endo--1,4-xylanases [8,17,21]. Such upregulation of rXylM activity is presumed to be due to the direct interaction of the enzyme with Triton X-100 or Tween 80, which may cause a change of the enzyme-substrate interaction [6]. In this study, rXylM was completely inactivated by Trp-directed modifiers, Hg2+ (1 mM) and Nbromosuccinimide (5 mM) when pre-incubated with the reactive chemicals for 10 min. These results agreed well with the finding that three Trp residues in the strictly conserved region of GH10 endo--1,4-xylanases are essential for enzyme-substrate interaction [24,25]. In premature XylM, the Trp129, Trp307, and Trp315 residues were predicted to play a crucial role in biocatalysis and substrate-binding of the enzyme. However, the suppression of rXylM activity by 1 mM Cu2+ was approximately 25% of its original biocatalytic activity, similar to that of Cellulosimicrobium sp. HY-13 GH10 endo--1,4-xylanase activity by the same cation [6,26]. It was also noted that the stimulation or inhibition of rXylM by metal ions (1 mM) including Ca2+, Ni2+, Zn2+, Mg2+, Mn2+, Sn2+, Ba2+, Co2+, and Fe2+ together with EDTA (5 mM) was only marginal. Moreover, insignificant alterations of rXylM activity by sulfhydryl reagents (5 mM), such as iodoacetamide, sodium azide, and N-ethylmaleimide, were comparable with the prior description of GH10 endo--1,4-xylanase suppression by the same reagents [21].

3.4. Substrate specificity

The substrate specificities of rXylM and rXylM△RICIN were investigated using various D-glucose-, Dmannose-, and D-xylose-based polysaccharides together with PNP-sugar derivatives. Of the tested substrates, rXylM was able to efficiently degrade -1,4-D-xylan polysaccharides with the following order: oat spelts xylan > beechwood xylan > birchwood xylan > wheat arabinoxylan. However, it did not display any detectable degradation activity toward xyloglucan, carboxymethylcellulose, locust bean gum, and PNP-sugar (D-cellobiose, D-glucose, Dmannose, and D-xylose) derivatives. These results suggest that rXylM is a true GH10 endo--1,4-xylanase lacking other glycoside hydrolase activities, which is distinct from the known bi-functional GH10 endo--1,4-xylanases [27,28]. The specific activities of rXylM for birchwood xylan and beechwood xylan were 162.2 U mg -1 and 180.5 11

U mg-1, respectively. In addition, the specific activity of rXylM for oat spelts xylan was 254.1 U mg-1, with a relatively low specific activity (76.6 U mg-1) for wheat arabinoxylan. The oat spelts xylan-degrading activity of rXylM was 3.5- and 2.9-fold higher than that (72.6 U mg-1) of Paenicillium oxalicum GH10 endo--1,4-xylanase [29] and that (87.3 U mg-1) of Streptomyces thermocarboxydus HY-15 GH10 endo--1,4-xylanase [21] for the same substrate, respectively. However, the oat spelts xylan-degrading activity of rXylM was evaluated to be approximately 71.8% of that (353.7 U mg-1) of C. kronotskyensis GH10 endo--1,4-xylanase [23]. Compared to rXylM, rXylM△RICIN showed markedly reduced degradation activity toward D-xylose-based water-soluble and insoluble polysaccharides even though, like rXylM, it could not hydrolyze structurally unrelated polysaccharides and PNP-sugar derivatives. In particular, rXylM△RICIN was found to efficiently decompose -1,4-D-xylan polysaccharides with the following order: beechwood xylan > oat spelts xylan > birchwood xylan > wheat arabinoxylan. The specific activities of rXylM△RICIN for birchwood xylan and beechwood xylan were determined to be 63.1 U mg-1 and 101.6 U mg-1, respectively, which were less than 56.3% of the rXylM activities for the same substrates. It should also be noted that the specific activity (32.2 U mg-1) of rXylM△RICIN for wheat arabinoxylan with a high water-insoluble fraction was approximately 2.4-fold lower than that (76.6 U mg-1) of rXylM for the same polysaccharide. Moreover, the ability of rXylM△RICIN to degrade oat spelts xylan with a high water-insoluble fraction was 3.4-fold less than that of rXylM to hydrolyze the same substrate. The results suggest that compared to rXylM, a significant decrease of the biocatalytic activity of rXylM△RICIN, especially for insoluble -1,4-D-xylan polysaccharides, might reflect the absence of a RICIN domain in the C-terminus region, which is expected to play a positive role in enzyme-substrate binding and biocatalysis [30]. Similarly, CBMs have been also shown to play a considerable role in breakdown of insoluble -1,4-D-xylans than soluble -1,4-D-xylans [23,31]. The kinetic parameters (Vmax, Km, Kcat, and Kcat/Km) for the enzymatic degradation of beechwood xylan, birchwood xylan, oat spelts xylan, and wheat arabinoxylan are shown in Table 1. Compared to rXylM△RICIN, rXylM displayed lower Km values for the same -1,4-D-xylan polysaccharides, indicative of higher substratebinding affinities. For example, the Km values of rXylM and rXylM△RICIN toward oat spelts xylan were 1.26 mg 12

mL-1 and 2.31 mg mL-1, respectively. In addition, rXylM showed the Km value of 1.39 mg mL-1 toward beechwood xylan but the Km value of rXylM△RICIN toward the same substrate was measured to be 1.98 mg mL-1. In contrast to the Km values of rXylM toward the evaluated -1,4-D-xylan polysaccharides, its Vmax, Kcat, and Kcat/Km values were significantly higher than those of rXylM△RICIN toward the same substrates. Specifically, the Vmax, Kcat, and Kcat/Km values of rXylM toward oat spelts xylan were 4.6-, 6.5-, and 11.9-fold higher, respectively, than those of rXylM△RICIN toward the same substrate. Likewise, the Vmax, Kcat, and Kcat/Km values of rXylM toward wheat arabinoxylan were 2.7-, 3.8-, and 5.2-fold higher, respectively, than those of rXylM△RICIN when the same substrate was used. These findings were in good agreement with the results that the specific activities of rXylM were higher than those of rXylM△RICIN for the same substrates. The results of HPLC analysis clearly revealed that a series of -1,4-D-xylooligosaccharides of X3 to X6 as well as birchwood xylan could be readily hydrolyzed by rXylM, even though X2 was highly resistant to the enzyme (Table 2). Specifically, rXylM decomposed birchwood xylan to X3 (58.9%) and X2 (37.1%) together with small amounts of X4 (2.6%) and X1 (1.4%) as the enzyme reaction was carried out for 6 h at 30 oC. In addition, the enzyme efficiently cleaved -1,4-D-xylooligomers of X4 to X6 to produce X3 (> 49%) as the major end product. However, the biocatalytic degradation of X3 appeared to proceed relatively slowly, yielding a mixture of X1 (0.8%), X2 (16.7%), and X3 (82.5%) under the given reaction conditions. The hydrolysis patterns of -1,4-Dxylooligosaccharides (X2-X6 ) and birchwood xylan by rXylM were relatively similar to those of the same substrates by rXylM△RICIN (data not shown). Up to this time, diverse GH10 endo--1,4-xylanases have been demonstrated as a retaining enzyme with notable transglycosylation activity to synthesize longer -1,4-Dxylooligomers from X3 or X4 [6,17,32]. Thus, it is interesting to note that rXylM did not exhibit any detectable transglycosylation activity in degradation reaction of -1,4-D-xylooligomers (X2-X6), similar to extracelluar GH10 endo--1,4-xylanases of Streptomyces mexicanus HY-14 [8] and C. kronotskyensis [23]. The inability of rXylM to produce longer -1,4-D-xylooligomers from the substrates also corresponded to the fact that it could not cleave PNP-cellobioside, indicative of the absence of transglycosylation activity. It has previously been reported that some GH10 endo--1,4-xylanases with notable transglycosylation activity display relatively high cleavage activity 13

against PNP-cellobioside [8,21,32]. Taken together, the substrate hydrolysis patterns of rXylM clearly indicate that the enzyme is an endo-type -1,4-xylanase without transglycosylation activity, which is capable of efficiently degrading -1,4-D-xylans and -1,4-D-xylooligomers greater than X3.

3.5. Binding affinity of rXylM and rXylM△RICIN to insoluble materials

Using diverse insoluble materials such as pentose- and hexose-based polysaccharides and lignin, substratebinding affinities of two GH10 endo--1,4-xylanases with distinct molecular structures and domain architectures have been recently investigated [17,21]. However, a study on substrate-binding capacity of a GH10 endo--1,4xylanase with a RICIN domain has not been documented to date. Therefore, to evaluate the role of a C-terminal RICIN domain in XylM in enzyme-substrate binding, we investigated the binding abilities of rXylM and rXylM△RICIN to various hydrophobic polymers with a unique microstructure. The results of the binding assay revealed that rXylM could be strongly bound to lignin, insoluble oat spelts xylan, Avicel, and ivory nut mannan, displaying high binding affinities (> 90%) (Fig. 6). However, the binding affinity of rXylM to chitin was found to be less than 60%. These substrate-binding patterns of rXylM were comparable to those of other characterized GH10 enzymes toward the same insoluble polymers. Previously, a GH10 endo--1,4-xylanase (rXylU) with a C-terminal CBM 2 domain from Streptomyces mexicanus HY-14 was reported to show relatively high binding capacities (> 85%) to chitin [21]. In addition, the binding affinities of Microbacterium trichothecenolyticum HY-17 endo--1,4xylanase (rXylH) consisting of a single catalytic GH10 domain to chitosan and chitin exceeded 85% [17]. Furthermore, rXylH [17] displayed only weak binding capacity (< 45%) to lignin compared to modular GH10 enzymes (rXylM and rXylU [21]). It is interesting to note that compared to rXylM, rXylM△RICIN exhibited relatively lower substrate-binding affinity to lignin, chitosan, chitin, insoluble oat spelts xylan, and ivory nut mannan, although its binding ability to Avicel was almost similar to that of rXylM to the same polysaccharide. The reduction of substrate-binding affinities caused by the deletion of a RICIN domain in XylM was very similar to that of substrate-binding affinities originated by the the removal of a CBM 2 domain in rXylU [21]. These findings 14

suggest that the RICIN domain in XylM might play a crucial role in the formation of enzyme-substrate complex, which promotes biocatalytic degradation of -1,4-D-xylan polysaccharides.

4. Conclusion

The extracellular, modular -1,4-D-xylan-depolymerizing enzyme (rXylM) from L. xylanilyticum HY-24 is a unique GH10 endo--1,4-xylanase with a RICIN domain as a substrate-binding motif that exhibits distinct characteristics in its primary structure, hydrolysis patterns of xylosic materials to generate X3 as the major product, and binding affinities to insoluble polymers. Compared to rXylM, the lower binding abilities and biocatalytic activities of rXylM△RICIN toward -1,4-D-xylan polysaccharides suggest that the RICIN domain plays a crucial role in enzyme-substrate binding and biocatalysis. Considering its ability to dominantly produce X2 and X3 from the degradation of xylosic materials, rXylM can be exploited as a potential candidate for the preparation of prebiotic 1,4-D-xylooligosaccharides that will promote the growth of probiotics in the intestines of herbivorous animals [33].

Acknowledgements

This work was supported by the Grant from the KRIBB Research Initiative Program (KGM2131723) and the Bio & Medical Technology Development Program (PRM0151712), Republic of Korea.

15

References [1] R.C. Kuhad, R. Gupta, A. Singh, Microbial cellulases and their industrial applications, Enzyme Res. 2011 (2011) 280696. [2] D. Shallom, Y. Shoham, Microbial hemicellulases, Curr. Opin. Microbiol. 6 (2003) 219-228. [3] M.L.T.M. Polizeli, A.C.S. Rizzatti, R. Monti, H.F. Terenzi, J.A. Jorge, D.S. Amorim, Xylanases from fungi: properties and industrial applications, Appl. Microbiol. Biotechnol. 67 (2005) 577-591. [4] V. Juturu, J.C. Wu, Microbial xylanases: engineering, production and industrial applications, Biotechnol. Adv. 30 (2012) 1219-1227. [5] S. Subramaniyan, P. Prema, Biotechnology for microbial xylanases: enzymology, molecular biology, and application, Crit. Rev. Biotechnol. 22 (2002) 33-64. [6] D.Y. Kim, M.K. Han, D.-S. Park, J.S. Lee, H.-W. Oh, D.-H. Shin, T.-S. Jeong, S.U. Kim, K.S. Bae, K.-H. Son, H.-Y. Park, Novel GH10 xylanase, with a fibronectin type 3 domain, from Cellulosimicrobium sp. strain HY13, a bacterium in the gut of Eisenia fetida, Appl. Environ. Microbiol. 75 (2009) 7275-7279. [7] J. Ni, G. Tokuda, Lignocellulose-degrading enzymes from termites and their symbiotic microbiota, Biotechnol. Adv. 31 (2013) 838-850. [8] D.Y. Kim, D.-H. Shin, S. Jung, J.S. Lee, H.-Y. Cho, K.S. Bae, C.-K. Sung, Y.H. Rhee, K.-H. Son, H.-Y. Park, Biocatalytic properties and substrate-binding ability of a modular GH10 -1,4-xylanase from an insectsymbiotic bacterium, Streptomyces mexicanus HY-14, J. Microbiol. 52 (2014) 863-870. [9] J. Zhou, H. Huang, K. Meng, P. Shi, Y. Wang, H. Luo, P. Yang, Y. Bai, Z. Zhou, B. Yao, Molecular and biochemical characterization of a novel xylanase from the symbiotic Sphingobacterium sp. TN19, Appl. Microbiol. Biotechnol. 85 (2009) 323-333. [10] D.Y. Kim, M.J. Lee, H.-Y. Cho, J.S. Lee, M.-H. Lee, C.W. Chung, D.-H. Shin, Y.H. Rhee, K.-H. Son, H.-Y. Park, Genetic and functional characterization of an extracellular modular GH6 endo--1,4-glucanase from an earthworm symbiont, Cellulosimicrobium funkei HY-13, Antonie van Leeuwenhoek 109 (2016) 1-12. [11] D.Y. Kim, C.W. Chung, H.-Y. Cho, Y.H. Rhee, D.-H. Shin, K.-H. Son, H.-Y. Park, Biocatalytic 16

characterization of an endo--1,4-mannanase produced by Paenibacillus sp. strain HY-8, Biotechnol. Lett. 39 (2017) 149-155. [12] E.D. Scully, S.M. Geib, K. Hoover, M. Tien, S.G. Tringe, K.W. Barry, T. Glavina del Rio, M. Chovatia, J.R. Herr, J.E. Carlson, Metagenomic profiling reveals lignocellulose degrading system in a microbial community associated with a wood-feeding beetle, PloS One 8 (2013) e73827. [13] F. Warnecke, P. Luginbühl, N. Ivanova, M. Ghassemian, T.H. Richardson, J.T. Stege, M. Cayouette, A.C. McHardy, G. Djordjevic, N. Aboushadi, R. Sorek, S.G. Tringe, M. Podar, H.G. Martin, V. Kunin, D. Dalevi, J. Madejska, E. Kirton, D. Platt, E. Szeto, A. Salamov, K. Barry, N. Mikhailova, N.C. Kyrpides, E.G. Matson, E.A. Ottesen, X. Zhang, M. Hernández, C. Murillo, L.G. Acosta, I. Rigoutsos, G. Tamayo, B.D. Green, C. Chang, E.J. Mathur, D.E. Robertson, P. Hugenholtz, J.R. Leadbetter, Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite, Nature 450 (2007) 560-565. [14] H. Kim, H.-W. Oh, J.-A. Kim, D.-S. Park, H.-M. Park, K.S. Bae, Luteimicrobium xylanilyticum sp. nov., isolated from the gut of a long-horned beetle, Massicus raddei, Int. J. Syst. Evol. Microbiol. 64 (2014) 14011405. [15] M.M. Bradford, A rapid and sensitive method for the quantitation of microorganism quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248-254. [16] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31 (1959) 426-428. [17] D.Y. Kim, D.-H. Shin, S. Jung, H. Kim, J.S. Lee, H.-Y. Cho, K.S. Bae, C.-K. Sung, Y.H. Rhee, K.-H. Son, H.Y. Park, Novel alkali-tolerant GH10 endo--1,4-xylanase with broad substrate specificity from Microbacterium trichothecenolyticum HY-17, a gut bacterium of the mole cricket Gryllotalpa orientalis, J. Microbiol. Biotechnol. 24 (2014) 943-953. [18] A.M. MacLeod, T. Lindhorst, S.G. Withers, R.A.J. Warren, The acid/base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is glutamic acid 127: evidence from detailed kinetic studies of mutants, Biochemistry 33 (1994) 6371-6376. 17

[19] N. Li, K. Meng, Y. Wang, P. Shi, H. Luo, Y. Bai, P. Yang, B. Yao, Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9, Appl. Microbiol. Biotechnol. 80 (2008) 231-240. [20] Y. Hu, G. Zhang, A. Li, J. Chen, L. Ma, Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach, Appl. Microbiol. Biotechnol. 80 (2008) 823-830. [21] D.Y. Kim, M.K. Han, H.-W. Oh, D.-S. Park, S.-J. Kim, S.-G. Lee, D.-H. Shin, K.-H. Son, K.S. Bae, H.-Y. Park, Catalytic properties of a GH10 endo-β-1,4-xylanase from Streptomyces thermocarboxydus HY-15 isolated from the gut of Eisenia fetida, J. Mol. Catal. B: Enzym. 62 (2010) 32-39. [22] H.Y. Song, H.K. Lim, D.R. Kim, K.I. Lee, I.T. Hwang, A new bi-modular endo--1,4-xylanase KRICT PX-3 from whole genome sequence of Paenibacillus terrae HPL-003, Enzyme Microb. Technol. 54 (2014) 1-7. [23] X. Jia, W. Qiao, W. Tian, X. Peng, S. Mi, H. Su, Y. Han, Biochemical characterization of extra- and intracellular endoxylanase from thermophilic bacterium Caldicellulosiruptor kronotskyensis, Sci. Rep. 2 (2016) 21672. [24] M. Roberge, F. Shareck, R. Morosoli, D. Kluepfel, C. Dupont, Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans, Protein Eng 12 (1999) 251-257. [25] G. Zolotnitsky, U. Cogan, N. Adir, V. Solomon, G. Shoham, Y. Shoham, Mapping glycoside hydrolase substrate subsites by isothermal titration calorimetry, Proc. Natl. Acad. Sci. USA 101 (2004) 11275-11280. [26] H. Shi, Y. Zhang, X. Li, Y. Huang, L. Wang, Y. Wang, H. Ding, F. Wang, A novel highly thermostable xylanase stimulated by Ca2+ from Thermotoga thermarum: cloning, expression and characterization, Biotechnol. Biofuels 6 (2013) 26. [27] J. Sermsathanaswadi, S. Baramee, C. Tachaapaikoon, P. Pason, K. Ratanakhanokchai, A. Kosugi, The family 22 carbohydrate-binding module of bifunctional xylanase/-glucanase Xyn10E from Paenibacillus curdlanolyticus B-6 has an important role in lignocellulose degradation, Enzyme Microb. Technol. 96 (2017) 75-84. [28] X. Xue, R. Wang, T. Tu, P. Shi, R. Ma, H. Luo, B. Yao, X. Su, The N-terminal GH0 domain of a 18

multimodular protein from Caldicellulosiruptor bescii is a versitile xylanase/-glucanase that can degrade crystalline cellulose, Appl. Environ. Microbiol. 81 (2015) 3823-3833. [29] H. Liao, S. Sun, P. Wang, W. Bi, S. Tan, Z. Wei, X. Mei, D. Liu, W. Raza, Q. Shen, Y. Xu, A new acidophilic endo--1,4-xylanase from Penicillium oxalicum: cloning, purification, and insights into the influence of metal ions on xylanase activity, J. Ind. Microbiol. Biotechnol. 41 (2014) 1071-1083. [30] Z. Fujimoto, A. Kuno, S. Kaneko, S. Yoshida, H. Kobayashi, I. Kusakabe, H. Mizuno, Crystal structure of Streptomyces olivaceoviridis E-86 -xylanase containing xylan-binding domain, J. Mol. Biol. 300 (2000) 575585. [31] M. Zhang, J.R. Chekan, D. Dodd, P.-Y. Hong, L. Radlinsk, V. Revindran, S.K. Nair, R.I. Mackie, I. Cann, Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes, Proc. Natl. Acad. Sci. USA 111 (2014) E3708-E3717. [32] D.Y. Kim, M.K. Han, H.-W. Oh, K.S. Bae, T.-S. Jeong, S.U. Kim, D.-H. Shin, I.-H. Kim, Y.H. Rhee, K.-H. Son, H.-Y. Park, Novel intracellular GH10 xylanase from Cohnella laeviribosi HY21: biocatalytic properties and alterations of substrate specificities by site-directed mutagenesis of Trp residues, Bioresour. Technol. 101 (2010) 8814-8821. [33] M.J. Vázquez, J.L. Alonso, H. Domínguez, J.C. Parajó, Xylooligosaccharides: manufacture and applications. Trends Food Sci. Technol. 11 (2000) 387-393.

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Figure captions

Fig. 1. Primary sequence alignment of L. xylanilyticum HY-24 GH10 endo--1,4-xylanase and its structural homologs. Sequences (GenBank accession numbers): Lxy, L. xylanilyticum HY-24 endo-β-1,4-xylanase (KY997451); Mlu, M. lupini endo-β-1,4-xylanase (WP_007459199); Agl, A. globisporus endo-β-1,4-xylanase (WP_020516021); Ame, A. mediterranei endo-β-1,4-xylanase (WP_013225148); Cfl, C. flavigena endo--1,4xylanase (WP_013115627). The identical and similar amino acids are shown by black and gray boxes, respectively. The predicted signal peptide is indicated by a black bar. The internal peptide sequences used in the design of degenerated PCR primers are marked by arrows. Highly conserved amino acid residues that play a critical role in biocatalysis are indicated by asterisks. GH10 and RICIN domains are outlined by solid and dotted lines, respectively.

Fig. 2. Structure-based sequence alignment of L. xylanilyticum HY-24 GH10 endo-β-1,4-xylanase and its structural homologs, which was prepared with ESPript 3.0 program. The first line indicates the secondary structure elements of Streptomyces lividans GH10 endo-β-1,4-xylanase (PDB code: 1E0V) that was used as a template. -Helices and -strands are represented as squiggles and arrows, respectively.  displays a 310-helix and strict -turns are marked by the letters TT. The disulfide-forming Cys residues are indicated by numbers pairwise. Sequences (GenBank accession numbers): Sli, S. lividans endo-β-1,4-xylanase (AAC26525); Lxy, L. xylanilyticum HY-24 endo-β-1,4xylanase (KY997451); Mlu, M. lupini endo-β-1,4-xylanase (WP_007459199); Agl, A. globisporus endo-β-1,4xylanase (WP_020516021); Ame, A. mediterranei endo-β-1,4-xylanase (WP_013225148).

Fig. 3. SDS-PAGE of the purified rXylM and rXylM△RICIN after affinity chromatography on HisTrap TM HP. Lane S, standard marker proteins; lane 1, rXylM; lane 2, rXylM△RICIN.

Fig. 4. Effect of pH on the endo-β-1,4-xylanase activity of rXylM (a) and rXylM△RICIN (b) and effect of pH on 20

the stability of rXylM (c) and rXylM△RICIN (d). The optimal pH of rXylM and rXylM△RICIN was assessed using the following buffers (50 mM): sodium citrate (●), sodium phosphate (○), Tris-HCl (▲), and glycine-NaOH (▽).

Fig. 5. Effect of temperature on the endo-β-1,4-xylanase activity (a) and stability (b) of rXylM (○) and rXylM△RICIN (●).

Fig. 6. Binding of rXylM (□) and rXylM△RICIN (■) to insoluble polymers.

21

22

23

24

25

26

27

Table 1 Kinetic parameters of rXylM and rXylM△RICIN determined using 0.2-1.5% of each substrate.

rXylM

rXylM△RICIN

Substrate Vmax

Km

kcat

kcat/Km

Vmax

Km

kcat

kcat/Km

(U mg-1)

(mg mL-1)

(s-1)

(mg-1 s-1 mL)

(U mg-1)

(mg mL-1)

(s-1)

(mg-1 s-1 mL)

Beechwood xylan

294.7

1.4

248.0

178.4

140.6

2.0

84.4

42.2

Birchwood xylan

263.5

1.5

221.8

153.0

80.8

2.5

48.5

19.4

Oat spelts xylan

463.1

1.3

389.8

309.4

100.1

2.3

60.1

26.1

Wheat arabinoxylan

100.7

2.8

84.8

29.9

37.1

3.9

22.3

5.7

Kinetic parameter values are the average of three replicates.

Table 2 LC analysis of the hydrolysis products of xylosic materials by rXylM. Composition (%)a of products formed by hydrolysis reaction Substrate X1

X2

X3

X4

X5

X2

0.3

99.7

X3

0.8

16.7

82.5

X4

1.9

34.9

49.2

14.0

X5

1.2

38.4

49.1

6.7

4.6

X6

1.2

29.8

56.4

10.9

1.7

Birchwood xylan

1.4

37.1

58.9

2.6

a

LC area%.

28