Metagenomic Mining and Functional Characterization of a Novel

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Biotechnology and Biological Transformations

Metagenomic mining and functional characterization of a novel KG51 bifunctional cellulase/hemicellulase from black goat rumen Kyung-Tai Lee, Sazzad Hossen Toushik, Jin-Young Baek, Ji-Eun Kim, Jin-Sung Lee, and Keun-Sung Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01449 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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Journal of Agricultural and Food Chemistry

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Metagenomic mining and functional characterization of a novel KG51 bifunctional

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cellulase/hemicellulase from black goat rumen

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Kyung-Tai Lee2 , Sazzad Hossen Toushik1, Jin-Young Baek1, Ji-Eun Kim1, Jin-Sung Lee,3* and

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Keun-Sung Kim 1*

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Korea

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Department of Food Science and Technology, Chung-Ang University, Ansung 456-756, South

Animal Genomics and Bioinformatics Division, National Institute of Animal Science, Rural

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Development Administration, Wanju 565-851, South Korea

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Department of Biological Sciences, Kyonggi University, Suwon 442-760, South Korea

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Abstract word count: 149

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Text word count (excluding abstract and references): 4,396

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Text word count (including abstract and references): 5,759

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Running title: Novel KG51 bifunctional cellulase/hemicellulase

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*Corresponding authors

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Jin-Sung Lee, [email protected]; Keun-Sung Kim, [email protected]

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ACS Paragon Plus Environment


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Abstract A novel KG51gene was isolated from a metagenomic library of Korean black goat rumen

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and its recombinant protein was characterized as a bifunctional enzyme (cellulase/hemicellulase).

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In silico sequence and domain analyses revealed that the KG51 gene encodes a novel

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carbohydrate-active enzyme that possesses a salad bowl-like shaped glycosyl hydrolase family 5

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(GH5) catalytic domain but, at best, 41% sequence identity with other homologous GH5 proteins.

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Enzymatic profiles (optimum pH values and temperatures, as well as pH and thermal stabilities)

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of the recombinant KG51 bifunctional enzyme were also determined. Based on the substrate

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specificity data, the KG51 enzyme exhibited relatively strong cellulase (endo-β-1,4-glucanase

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[EC 3.2.1.4]) and hemicellulase (mannan endo-β-1,4-mannosidase [EC 3.2.1.78] and endo-β-1,4-

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xylanase [EC 3.2.1.8]) activities, but no exo-β-1,4-glucanase (EC 3.2.1.74), exo-β-1,4-glucan

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cellobiohydrolase (EC 3.2.1.91), and exo-1,4-β-xylosidase (EC 3.2.1.37) activities. Finally, the

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potential industrial applicability of the KG51 enzyme was tested in the preparation of prebiotic

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konjac glucomannan hydrolysates.

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Keywords: rumen, cellulase, hemicellulase, bifunctional, glycosyl hydrolase family 5

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Introduction Lignocellulose is the structural building block of woody and non-woody plants and a

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composite of the carbohydrate polymers cellulose (38–50%), hemicellulose (23–32%), and

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pectin (35–40%) and the non-carbohydrate polymer lignin (15–25%) depending on the plant

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variety 1. Particularly, cellulose and hemicellulose are major constituents of the cell wall of

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vascular plants 2 with the same β-1,4-glycosidic bonds in their backbone 3. Cellulose can be

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degraded into glucose and short cellodextrins through hydrolysis by three classes of cellulases,

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which are endo-β-1,4-glucanase (EC 3.2.1.4), exo-1,4-β-cellobiohydrolase (EC 3.2.1.91), and β-

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glucosidase (EC 3.2.1.21) 2. The two major hemicellulosic components are hetero-1,4-β-D-xylans

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and hetero-1,4-β-D-mannans in hardwood and softwood plants, respectively 4. Complete

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degradation of hemicellulose in hardwood plants requires the synergistic action of the

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hemicellulases endo-β-1,4-xylanase (EC 3.2.1.8), endo-1,4- β-mannanase (EC 3.2.1.78), exo-1,4-

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β-xylosidase (EC 3.2.1.37), α-glucuronidase (EC 3.2.1.139), α-arabinofuranosidase (EC

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3.2.1.55), and acetylalkylglycerol acetylhydrolase (EC 3.1.1.71) 1. Among the hemicellulases,

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endo-β-1,4-xylanase and exo-1,4- β-xylosidase play crucial roles in the hydrolysis of xylan, the

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backbone of hemicellulose 2. In addition to endo-β-1,4-xylanase, endo-1,4- β-mannanase plays a

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decisive role in the hydrolysis of hemicellulose in softwood plants 4. Considering the

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compatibility of cellulase and hemicellulase, using both enzymes simultaneously could

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efficiently enhance the quality of wheat flour and rapidly digestible animal feed products during

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processing 1. Additionally, both cellulase and hemicellulase can be efficiently used to produce

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effective prebiotics and stimulate the growth of lactic acid bacteria in the human or animal gut 5.

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Thus, a bifunctional cellulase/hemicellulase enzyme could be a good choice for such processes.

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Natural feed sources of ruminants are primarily substances of plant origin, mostly 3



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comprising lignocellulosic biomass. However, ruminants depend on the symbiotic microbes

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residing in their gastrointestinal (GI) tracts, comprising anaerobic bacteria, fungi, methanogenic

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archaea, and protozoa that synthesize various lignocellulolytic enzymes 6, to digest the feed 7.

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Especially, the rumen microbial communities mainly rapidly and completely degrade cellulosic

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and hemicellulosic feeds 8. However, the microbial communities have been reported to be highly

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diverse and contain many previously unidentified microorganisms 9.

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Many screening experiments have been conducted to isolate and characterize

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lignocellulolytic enzymes from various sources 10. Previously, culture-dependent screening

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approaches were used to isolate those enzymes, but these traditional isolation approaches could

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not screen for more than 99% of the lignocellulolytic microbial communities of rumens because

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they were unculturable 11. Metagenomics-based approaches can be effectively used to overcome

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the technical limitations of traditional cultivation-based approaches when novel functional genes

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are screened from the unculturable microbial communities of rumens 6. Among the different

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rumens of various ruminants, this study focused on Korean goat rumens because of the unique

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digestibility of lignocellulosic biomasses.

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In our previous study, a fosmid metagenomic library was constructed from rumen

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microbial communities of black goats 12. A high abundance of clones with carboxymethyl

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cellulase (CMCase) activity was identified, suggesting that this metagenomic library was a rich

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reservoir of various cellulase genes 12. Hence, in this study, we used activity-based screening to

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isolate a novel gene (KG51) from this library, which was subjected to in silico characterization of

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the sequence and domain and subsequently expressed in Escherichia coli. The enzymatic

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characteristics of the recombinant KG51 protein were analyzed using various cellulosic and

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hemicellulosic substrates under different pH and temperature ranges. To test the potential 4


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industrial application of the recombinant KG51 enzyme, it was used to prepare a konjac

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glucomannan hydrolysate (KGMH), which was investigated for prebiotic effects in vitro using

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Lactobacillus acidophilus KCTC 2182.

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Materials and methods Construction and functional screening of black goat rumen metagenomic fosmid library Three 18-month-old Korean black goats were carefully fed rice straw and mineral

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supplements for the 30-day period preceding the experiment to maximize lignocellulolytic

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adaptation of microorganisms in their rumen. Metagenomic DNA was extracted from their rumen,

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and a black goat rumen metagenomic fosmid library was constructed as described previously 12.

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Each clone from the constructed fosmid library was screened for CMCase activity as

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described previously 12. The activity-based metagenomic screening identified a single fosmid

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clone (Lq007G03) that formed one of the largest clear halos, and the clone was isolated for

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further experiment.

98 99 100

Shotgun sequencing and de novo assembly of enzyme-positive fosmid clone Bacteria carrying the lignocellulolytic fosmid clone (Lq007G03) were grown in LB

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medium containing 25 µg/mL chloramphenicol for 20 h at 37°C, and the fosmid DNA was

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extracted from the enzyme-positive clone (Lq007G03) as described previously 12. Extracted

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DNA samples were pooled together to prepare the sample for shotgun sequencing using two

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approaches, conventional Sanger sequencing and pyrosequencing. The sequence data were 5



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obtained using shotgun sequencing as described previously, 12 and were subsequently assembled

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using the Phred and Phrap software programs for Sanger sequencing (University of Washington,

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Seattle, WA, USA) and the GS De Novo Assembler, version 2.7 (Roche, Basel, Switzerland) for

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pyrosequencing as described in a previous study 12.

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In silico sequence and domain analyses of enzyme-positive fosmid clone A full metagenomics-insert-DNA sequence was constructed by combining all contigs

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from the conventional Sanger sequencing data and the assembled pyrosequencing data as

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described previously 12. Potential open reading frames (ORFs) were predicted from the

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assembled contigs using the online server MetaGeneMark 13 and the developed heuristic

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approaches 14. Functional annotations of the ORFs were carried out by searching Pfam domains

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on the Pfam website (http://pfam.xfam.org) 15. Amino acid sequences were predicted from the

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nucleotide sequences of the enzyme-positive fosmid clone (Lq007G03). The nucleotide sequence

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of the putative KG51 gene was analyzed using an in silico program

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(http://insilico.ehu.es/translate). A BlastP search of the National Center for Biotechnology

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Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) database was used to determine the

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amino acid sequence similarities with other proteins derived from the previously identified ORFs.

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Multiple alignments of the amino acid sequences deduced from the ORFs and the sequences of

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other proteins were performed using the Clustal Omega program

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(http://www.ebi.ac.uk/Tools/msa/clustalo). Conserved domains in the ORFs were analyzed using

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the ExPasy PROSITE database search (http://prosite.expasy.org). The physical and chemical

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parameters of the KG51 protein were analyzed using the ProtParam program 6


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(http://web.expasy.org/protparam). The SignalP (http://www.cbs.dtu.dk/services/SignalP) and

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HMMTOP 2.0 (http://www.enzim.hu/hmmtop) programs were used to analyze the signal peptide

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cleavage sites within the N-terminal region of the ORFs and transmembrane regions, respectively.

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The tertiary (three-dimensional [3D]) structure of the KG51 protein was predicted using I-

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TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER) online platform based on homology

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modeling, LOMETS program (multiple threading template alignments), and iterative TASSER

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assembly simulations program 16. The predicted 3D model of the KG51 protein was further

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visualized and analyzed using the SPDBV 4.0.4 (http://swissmodel.expasy.org). The Verify3D

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program (http://services.mbi.ucla.edu/Verify_3D) was used to obtain information about protein

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fitness and validate the predicted KG51 protein model.

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Expression and purification of recombinant KG51 protein The putative lignocellulolytic KG51 gene was amplified using polymerase chain reaction

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(PCR) of the enzyme-positive fosmid clone (Lq007G03). The amplified DNA was then ligated

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into the expression vector pET-24a(+) carrying the C-terminal His6-tag (Novagen, Darmstadt,

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Germany) and transformed into E. coli Rosetta-gami (Novagen). Additionally, the bifunctional

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(cellulase and xylanase) activity of the recombinant enzyme was assayed using the agar plate

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method with carboxymethyl cellulose (CMC) or beechwood xylan (Sigma-Aldrich, St. Louis,

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MO, USA) as described by Geng et al.17. The recombinant KG51 protein was overexpressed in E.

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coli transformants and purified as previously described 12. To confirm the enzymatic activity, a

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2.0-µL aliquot of purified protein was inoculated into the agar plates containing CMC. The

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recombinant KG51 protein was analyzed using sodium dodecyl sulfate-polyacrylamide gel 7



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electrophoresis (SDS-PAGE) as described by Laemmli 18. Protein concentrations were

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determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). For zymogram

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analysis of the recombinant KG51 protein, the purified protein was analyzed using a 10% SDS-

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PAGE gel containing 0.5% CMC and 0.5% beechwood xylan for cellulase and xylanase,

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respectively, in the resolving gel as described previously 19.

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Enzymatic assays of recombinant KG51 enzyme The CMC and beechwood xylan substrates were used to analyze the activity of cellulase

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(CMCase) and xylanase, respectively. Using standards (glucose for CMCase or xylose for

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xylanase) under the assay conditions, the amount of enzyme releasing 1 µmol of reducing sugar

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in 1 min was defined as 1 unit (U) of enzymatic activity and the number of activity units per

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milliliter divided by the protein concentration (mg/mL) was the specific activity. Enzymatic

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activity was quantified by measuring the release of reduced sugar using 1% 3,5-dinitrosalicylic

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(DNS) reagent as described by Miller 20. The optimum pH and temperature conditions of the

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purified recombinant KG51 enzyme were determined for its cellulase and xylanase activities.

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Cellulase activity was measured by incubating 10 µL of the enzyme with 1% (w/v) CMC in 200

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µL of 50 mM sodium acetate buffer (pH 5.0) at 40°C for 15 min. Xylanase activity was

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measured by incubating 10 µL of the enzyme with 1% (w/v) beechwood xylan in 200 µL of 50

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mM sodium acetate buffer (pH 5.0) at 50°C for 10 min. The effects of pH and temperature on the

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cellulase and xylanase activities of KG51 enzyme were further examined. The optimal pH range

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was restricted in 50 mM sodium acetate buffer (pH 4.0–6.0), 50 mM sodium phosphate buffer

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(pH 6.0–8.0), and 50 mM Tris-HCl buffer (pH 8.0–10.0). The enzymatic reaction cocktails were 8


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incubated at different reaction temperatures ranging from 20 to 70°C for each activity to

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determine its optimum reaction temperature. The thermal stability of the KG51 enzyme was

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assessed by measuring the residual enzyme activity after pre-incubation for 1 h in 50 mM

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sodium acetate buffer at pH 5.0 for cellulase and xylanase activities at different reaction

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temperatures. The pH stability of the KG51 enzyme was also assessed after pre-incubation at

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4°C for 24 h in buffers with pH values ranging from 4.0 to 10.0. Substrate specificity was

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evaluated using various substrates including CMC, β-mannan, beechwood xylan, laminarin,

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avicel, barley glucan, larchwood xylan, p-nitrophenyl-β-glucopyranoside (pNPG), p-nitrophenyl-

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β-cellobiose (pNPC), and p-nitrophenyl-β-D-xylopyranoside (pNPX; all Sigma-Aldrich). The

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enzymatic activity of each substrate was determined by measuring the absorbance at 540 or 400

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nm for released reducing sugar or p-nitrophenol (pNP), respectively as described by Marques et

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al.21. All enzyme assays were carried out in triplicate.

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Preparation and prebiotic evaluation of KGMHs The KGM flour (Megazyme, Chicago, IL, USA) was dispersed (1 % w/v) in 50 mM

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sodium acetate buffer (pH 5.0) with the purified recombinant KG51 enzyme (0.3 U). The sample

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was incubated in a water bath at 50°C for 18 h. After incubation, the sample was heated for 15

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min to stop the enzymatic reaction. The enzyme hydrolysis was followed by the analysis of the

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increase in reducing sugars. The concentration of reducing sugars was determined using the DNS

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method as described previously 22. Mono-, di-, and oligosaccharides with desired degree of

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polymerization (DP) produced in the KGMHs were quantified using HPLC (Waters, Milford,

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MA, USA). After enzymatic reaction, the hydrolysates were filtered through 0.22 µm filter prior 9



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to injecting a sample volume of 10 µL into the HPLC column (Shodex SH-1011 column (7 µm,

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8.0 × 300 mm; Waters)). The column was maintained at 50°C and eluted with 0.01 N H2SO4 at a

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flow rate of 1 mL/min. Eluates were detected using a Waters 410 refractive index detector. Data

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acquisition and management were performed using Waters Empower™ Build 1154

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chromatography data software. Calibration standards for seven representative saccharides

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(Megazyme, Chicago, IL, USA), with an average DP of one (monosaccharide; DP1) to six

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(hexasaccharide; DP6) based upon their retention time, were run as above at 1 mg/mL for the

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monosaccharides (DP1) glucose and mannose, disaccharide (DP2) maltose, trisaccharide (DP3)

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maltotriose, tetrasaccharide (DP4) maltotetraose, pentasaccharide (DP5) maltopentaose, and

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hexasaccharide (DP6) maltohexaose. The concentration of each saccharide in mixture was then

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calculated as described previously 23.

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The prebiotic properties of the KGMHs prepared with the KG51 enzyme were

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determined using the method of Sutherland et al.24. The probiotic Lactobacillus acidophilus

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KCTC 2182 was used to evaluate the prebiotic properties of the KGMHs and grown in De Man,

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Rogosa, and Sharpe (MRS; BD, Franklin Lakes, NJ, USA) broth at 37°C for 48 h. In addition,

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two modified MRS broth media containing either 0.1% w/v KGM or KGMHs were used to

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evaluate the prebiotic properties. After incubation, the increased bacterial cell numbers in all the

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three different MRS broth media were determined using a quantitative reverse transcription

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(qRT)-PCR method. A pair of primers Lab_AF2 (5´-CGGTAATACGTAGGTGGCAA-3´) and

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Lab_BR1 (5- GCTACACATGGAGTTCCACT-3´) were designed based on the sequences of 16S

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rRNA gene from the L. acidophilus KCTC 2182. This primer pair amplified a 162-bp internal

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region of the 16S rRNA gene. The amplification and detection were performed with 20-µL

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reaction mixtures containing 10 pmol of each primer, 2 µL DNA template, and 10 µL 2× Real10


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time PCR Master Mix, SYBR Green I (Biofact Co. Ltd., Daejeon, South Korea) using a

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StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).

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Amplification conditions were 95°C for 10 min, followed by 40 cycles at 95°C for 30 s, 55°C for

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1 min, and 72°C for 1 min. The baseline and cycle threshold (CT) were automatically calculated,

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and a melting curve analysis was performed using the same equipment after the qRT-PCR was

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completed.

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Deposition in GenBank database The sequence of the enzyme-positive fosmid clone Lq007G03 including the complete

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ORF of the KG51 gene (a novel GH5 bifunctional cellulase/hemicellulase gene) has been

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deposited in the GenBank database under accession number KJ631403.1.

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Statistical analysis

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All experiments were carried out in triplicate and analyzed using the statistical package

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for the social sciences (SPSS) v23.0 for windows program (SPSS Inc., Chicago, IL, USA). All

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the data were presented as the means ± standard deviation (SD) of three independent experiments

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(n = 3). A paired t-test was used to determine the significance of the data and values with p
30% and 60% of CMCase and xylanase activities,

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respectively at pH 5.0–10.0 (Fig. 5C). The enzyme retained > 40% CMCase and xylanase

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activities at 20–50 °C and 20–40 °C, respectively (Fig. 5D). The KG51 enzyme exhibited

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considerable stability at the optimized temperatures and pH values for both activities. The

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purified recombinant bifunctional KG51enzyme activity was also assayed against various

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substrates (Table 2). The recombinant KG51 exhibited higher enzymatic activity on substrates

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with β-1,4 linkages, CMC (3.79 U/mg), β-mannan (3.28 U/mg), and beechwood xylan (2.63

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U/mg) than it did on substrates with mixed β-1,3/6 linkages such as laminarin (1.46 U/mg) or

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mixed β-1,3/4 linkages such as barley glucan (1.13 U/mg). Additionally, the recombinant

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bifunctional KG51enzyme exhibited trace activity towards the substrate larchwood xylan and did

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not hydrolyze the three synthetic substrates, pNPG, pNPC, and pNPX.

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Preparation and prebiotic evaluation of KGMHs The KGMHs prepared with the bifunctional KG51 enzyme were evaluated for their prebiotic activity on a probiotic strain L. acidophilus KCTC 2182. The reducing sugars in the 14


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KGMHs increased from 1.60 mg/mL to 5.85 mg/mL after the enzymatic preparation. Degree of

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polymerization (DP) profiles of 0.1% KGM and 0.1% KGMHs prepared with the bifunctional

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KG51 enzyme were determined by HPLC (Table 3). After 18 h hydrolysis, approximately 89%

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polysaccharides in the KGM flour solution were hydrolyzed into oligosaccharides of DP 2–6,

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and only a small amount of monosaccharide (8.66% of the oligosaccharides) was produced. The

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KG51 enzyme produced oligosaccharides with DP4 (27%) and DP5 (26%) as the two

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predominant hydrolysates in the KGMHs.

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Evaluation of the prebiotic evolution of the KGMHs revealed that the cell number

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increase of the probiotic strain in the MRS containing 0.1% KGMHs was 4.39 log cfu/mL after a

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48-h incubation, while that of the MRS only and MRS containing 0.1% KGM was 3.79 and 2.97

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log cfu/mL, respectively (Table 4). The KGMHs but not the KGM promoted the in vitro growth

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of the probiotic strain. Therefore, the in vitro fermentation results suggest that the KGMHs

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prepared with the KG51 enzyme treatment may have prebiotic properties.

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Discussion Cellulose and hemicellulose carbohydrates in dietary fibers are two major components

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covalently interlinked by lignin, the only non-carbohydrate component 2. The two carbohydrates

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must be enzymatically degraded to be used as carbon and energy sources in the rumen of

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herbivorous animals. Therefore, metagenomic approaches have been successfully used to screen

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a variety of carbohydrate-active enzymes (CAZymes) from the rumen of these animals 8.

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However, this is the first metagenomic-based attempt to isolate and characterize a novel

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bifunctional CAZyme (cellulase/hemicellulase) from unidentified dietary fiber degrading

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bacteria residing in the rumen of black goats. The construction and activity-based screening of a 15



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black goat rumen-derived metagenomic fosmid library subsequently identified 155 independent

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cellulolytic fosmid clones from the rumen. One of the clones contained the KG51 gene, and its

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recombinant product showed higher degrading activities on the β-1,4 bonds of both cellulose and

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hemicellulose carbohydrates than on the other bond types.

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The KG51 protein identified in this study shared less than 41% identity with the signature

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sequence of other homologous GH5 proteins. Despite the low degree of conservation in their

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amino acid sequences, all the GH5 enzymes share a consensus signature sequence of [LIV] –

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[LIVMFYWGA](2) – [DNEQG] – [LIVMGST] –{SENR}–N–E– [PV] – [RHDNSTLIVFY] 25.

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GH5, historically known as “cellulase family A”, is presently one of the largest and most diverse

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GH families and currently has 53 subfamilies. To date, the amino acid sequences of more than

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10,000 GH5 enzymes have been reported in the CAZy database, and 21 of them have been

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experimentally characterized and assigned an EC number. GH5 enzymes have various substrate

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specificities such as endo-β-1,4-glucanase/cellulase, endo-β-1,4-xylanase, β-mannosidase,

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mannan endo-β-1,4-mannosidase, cellulose β-1,4-cellobiosidase, licheninase, or chitosanase

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(CAZy database [http://www.cazy.org/]). The bioinformatics analysis in this study showed that

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the KG51 protein possesses only one known GH5 catalytic domain, but the recombinant KG51

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enzyme hydrolyzed both cellulose and hemicellulose substrates and, thereby acted as a

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bifunctional enzyme. This observation suggests that the activity of the KG51 bifunctional

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enzyme differs from that of other classical bifunctional enzymes 3, 26-27.

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The advent of advanced computer programming algorithms has enabled the prediction of

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the 3D structures of proteins and their functions based on their primary structures 16. The X-ray

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diffraction studies of the protein crystals showed that GH5 proteins maintain a catalytic 8-fold

350

(β/α)-barrel structure, usually forming a salad bowl-like shaped catalytic domain 28. In addition, 16


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GH5 CAZymes commonly have at least a conserved glutamic acid residue, which may act as a

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catalytic proton donor for the hydrolysis of cellulose substrates 28. The predicted 3D structure of

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the KG51 protein indicated that its eight parallel β-sheets are encircled by eight α-helixes,

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shaping the outer edge of the salad bowl. KG51 protein also has two putative catalytic residues

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as the proton donor (Glu200) and the nucleophile (Glu330) in its conserved GH5 sequence. The

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functional role of the two catalytic amino acid residues conserved in all members of the GH5

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was described recently 35. All the in silico structural data collected for the KG51 protein in this

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study strongly support this protein as a member of GH5.

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The substrate specificity of the recombinant KG51 enzyme towards 10 different

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substrates was determined in this study. Based on the data obtained in this study, the KG51

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enzyme exhibited relatively strong cellulase (endo-β-1,4-glucanase [EC 3.2.1.4]) and

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hemicellulase (mannan endo-β-1,4-mannosidase [EC 3.2.1.78] and endo-β-1,4-xylanase [EC

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3.2.1.8]) activities, but no exo-β-1,4-glucanase (EC 3.2.1.74), exo-β-1,4-glucan

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cellobiohydrolase (EC 3.2.1.91), and exo-1,4-β-xylosidase (EC 3.2.1.37) activities. Consequently,

365

the KG51 enzyme likely catalyzes the random hydrolysis of internal β-1,4-linked glycosidic

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bonds in cellulose and hemicellulose carbohydrates.

367

KGM is a hemicellulose derived from the tuber of the Amorphophallus konjac plant 29.

368

KGM is composed of a copolymer chain of 1,4-linked β-D-glucose and β-D-mannose units with

369

acetyl groups attached at a molar ratio of 1.6:1, having the molecular weight of KGM fiber

370

ranging from 500 to 2,000 kDa 5. A previous study comparatively evaluated the prebiotic effects

371

of KGM and acid hydrolyzed KGMHs with various DP on either lactobacilli or bifidobacteria,

372

which are normal components of the healthy human intestinal microflora, and demonstrated the

373

beneficial effects of the KGMHs with a lower DP 30. Particularly, glucomannooligosaccharides 17



Page 18 of 36

374

with a DP of 5 were reported to exert a greater prebiotic effect than those with a DP of 10 31.

375

Additionally, enzymatically hydrolyzed KGMHs with lower DPs have been reported to

376

efficiently enhance the growth of lactobacilli and bifidobacteria 32. Therefore, in this study, the

377

recombinant KG51 enzyme was used to hydrolyze KGM to produce low-molecular-weight

378

KGMHs and their prebiotic potential to promote the growth of a probiotic L. acidophilus strain

379

was subsequently examined. The present results indicate that the KG51 enzyme produced the

380

prebiotic KGMHs with DP of 2-6, which supported the growth of a probiotic Lactobacillus

381

species and the monoculture fermentations of the KGM or the KGMHs by the probiotic

382

Lactobacillus species demonstrated the potential applicability of the KG51 bifunctional enzyme

383

in preparing prebiotic KGMHs. In addition, the KG51 bifunctional enzyme has numerous

384

potential applications in relevant industries to improve the extraction and clarification of fruit

385

and vegetable juices, enrich soluble dietary fibers, increase digestibility of animal feeds, upgrade

386

bio-bleaching processes in the pulp and paper industry, enhance bio-polishing processes in the

387

textile industry, and produce second generation biofuels 2, 33-34.

388

In conclusion, a novel KG51 gene was isolated from a metagenomic library constructed

389

from the black goat rumens and its recombinant KG51 enzyme was functionally characterized. In

390

silico sequence and domain analyses of the KG51 gene and its protein were conducted. The

391

optimum reaction conditions and enzymatic stabilities of the KG51 enzyme were determined. In

392

addition, the substrate specificity of the KG51 enzyme towards 10 different substrates was

393

determined. Finally, its industrial application potential was demonstrated in the preparation of

394

prebiotic KGMHs.

395 396

Abbreviations Used 18


Page 19 of 36

397


CAZy, carbohydrate-active enzymes database; CAZymes, carbohydrate-active enzymes;

398

CMC, carboxymethyl cellulose; CMCase, carboxymethyl cellulase; DNA, deoxyribonucleic

399

acid; DNS, 3,5-dinitrosalicylic; DP, degree of polymerization; GH5, glycosyl hydrolase family 5;

400

GI, gastrointestinal; HPLC, high performance liquid chromatography; KCTC, Korean collection

401

for type cultures; KGM, konjac glucomannan; KGMH, konjac glucomannan hydrolysate; LB,

402

Luria-Bertani; MRS, De Man, Rogosa, and Sharpe; NCBI, National Center for Biotechnology

403

Information; ORF, open reading frames; PCR, polymerase chain reaction; pI, isoelectric point;

404

pNP, p-nitrophenol; pNPC, p-nitrophenyl-β-cellobiose; pNPG, p-nitrophenyl-β-glucopyranoside;

405

pNPX, p-nitrophenyl-β-D-xylopyranoside; qRT-PCR, quantitative reverse transcription-

406

polymerase chain reaction; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate-

407

polyacrylamide gel electrophoresis; SPSS, statistical package for the social sciences; 3D, three-

408

dimensional.

409 410

Author Information

411

Funding sources

412

This work was carried out with the support of the “Cooperative Research Programs for

413

Agriculture Science and Technology Development (Project Nos. PJ006649, PJ008701 and

414

PJ011163)”, Rural Development Administration, Republic of Korea. J.-S. Lee (for in silico

415

experiment design) and K.-S. Kim (for other experiment design) are co-corresponding authors of

416

this work.

417 418 419

Conflict of interest statement The authors declare no competing financial interest. 19



420

References

421

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422

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2. Toushik, S. H.; Lee, K.-T.; Lee, J.-S.; Kim, K.-S. Functional applications of lignocellulolytic

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xylanase/endoglucanase from yak rumen microorganisms. Appl. Microbiol. Biotechnol. 2011,

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5. Zhang, C.; Chen, J.-D.; Yang, F.-Q. Konjac glucomannan, a promising polysaccharide for OCDDS. Carbohydr. Polym. 2014, 104, 175-181. 6. Wang, L.; Xu, Q.; Kong, F.; Yang, Y.; Wu, D.; Mishra, S.; Li, Y. Exploring the goat rumen microbiome from seven days to two years. PloS One 2016, 11 (5), e0154354. 7. Ferrer, M.; Ghazi, A.; Beloqui, A.; Vieites, J. M.; López-Cortés, N.; Marín-Navarro, J.;

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20. Miller, G. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal.

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hydrolase family 1. Biochem. J. 2003, 370 (3), 793-804.

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23. Zang, H.; Xie, S.; Wu, H.; Wang, W.; Shao, X.; Wu, L.; Rajer, F. U.; Gao, X. A novel

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thermostable GH5_7 β-mannanase from Bacillus pumilus GBSW19 and its application in

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in Escherichia coli. Anim. Biotechnol. 2012, 23 (4), 261-277.

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26. Han, Y.; Dodd, D.; Hespen, C. W.; Ohene-Adjei, S.; Schroeder, C. M.; Mackie, R. I.; Cann, I.

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K. Comparative analyses of two thermophilic enzymes exhibiting both β-1,4 mannosidic and

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β-1,4 glucosidic cleavage activities from Caldanaerobius polysaccharolyticus. J. Bacteriol.

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2010, 192 (16), 4111-4121.

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27. Rashamuse, K. J.; Visser, D. F.; Hennessy, F.; Kemp, J.; Roux-van der Merwe, M. P.;

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Badenhorst, J.; Ronneburg, T.; Francis-Pope, R.; Brady, D. Characterisation of two

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bifunctional cellulase-xylanase enzymes isolated from a bovine rumen metagenome library.

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Curr. Microbiol. 2013, 66 (2), 145-151.

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28. Naas, A. E.; MacKenzie, A.; Dalhus, B.; Eijsink, V.; Pope, P. Structural features of a

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bacteroidetes-affiliated cellulase linked with a polysaccharide utilization locus. Sci. Rep. 2015,

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29. Liu, J.; Xu, Q.; Zhang, J.; Zhou, X.; Lyu, F.; Zhao, P.; Ding, Y. Preparation, composition

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analysis and antioxidant activities of konjac oligo-glucomannan. Carbohydr. Polym. 2015, 130,

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30. Chen, H. L.; Fan, Y. H.; Chen, M. E.; Chan, Y. Unhydrolyzed and hydrolyzed konjac

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glucomannans modulated cecal and fecal microflora in Balb/c mice. Nutrition 2005, 21 (10),

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31. Chen, J.; Liu, D.; Shi, B.; Wang, H.; Cheng, Y.; Zhang, W. Optimization of hydrolysis conditions for the production of glucomanno-oligosaccharides from konjac using β-mannanase 23



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by response surface methodology. Carbohydr. Polym. 2013, 93 (1), 81-88. 32. Gómez, B.; Míguez, B.; Yáñez, R,; Alonso, J. L. Manufacture and Properties of

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Glucomannans and Glucomannooligosaccharides Derived from Konjac and Other Sources. J.

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Agric. Food Chem. 2017, 65 (10), 2019-2031

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33. Xu, Q. H.; Wang, Y. P.; Qin, M. H.; Fu, Y. J.; Li, Z. Q.; Zhang, F. S.; Li, J. H. Fiber surface

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characterization of old newsprint pulp deinked by combining hemicellulase with laccase-

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mediator system. Bioresour. Technol. 2011, 102 (11), 6536-6540.

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34. Margeot, A.; Hahn-Hagerdal, B.; Edlund, M.; Slade, R.; Monot, F. New improvements for lignocellulosic ethanol. Curr. Opin. Biotechnol. 2009, 20 (3), 372-380. 35. Dadheech, T.; Shah, R.; Pandit, R.; Hinsu, A.; Chauhan, P.S.; Jakhesara, S.; Kunjadiya, A.;

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Rank, D.; Joshi, C. Cloning, molecular modeling and characterization of acidic cellulase from

523

buffalo rumen and its applicability in saccharification of lignocellulosic biomass. Int. J. Biol.

524

Macromol. 2018, 113, 73-81.

24


Page 25 of 36


525

Figure Legends

526

Figure 1. Multiple alignment of GH5 domain of KG51 enzyme with those of five other

527

homologous GH5 proteins. Signature sequence and catalytic residues of GH5 domain are

528

indicated in yellow and red boxes, respectively. Identical amino acid residues among six proteins

529

are shown in gray boxes. WP_005357523.1, CBL34359.1, CDE80894.1, KNY25463.1, and

530

WP_036943060.1 are GenBank accession numbers for Eubacterium siraeum hypothetical

531

protein, E. siraeum V10Sc8a endoglucanase, Ruminococcus sp. CAG:353 endoglucanase,

532

Pseudobacteroides cellulosolvens ATCC 35603 = DSM 2933 GH5 protein, and P. cellulosolvens

533

glycoside hydrolase, respectively.

534 535

Figure 2. In silico analysis of KG51gene product. Domain architecture of KG51 protein shows

536

its GH5 domain.

537 538

Figure 3. Predicted tertiary (three-dimensional [3D]) structure of KG51 protein. (A) Top, (B)

539

bottom, and (C) lateral views. Unique regions are depicted in different colors: α-helix (red), β-

540

sheet (yellow), random coil (blue), and putative catalytic residues (green).

541 542

Figure 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of

543

recombinant bifunctional KG51 enzyme overexpressed in Escherichia coli. Lane M, protein

544

molecular weight markers; lane 1, total cell-free extracts before induction; lane 2, soluble

545

fraction after induction; lane 3, purified recombinant KG51 enzyme; lane 4, zymogram analysis

546

of KG51 enzyme on 0.5% CMC-complemented PAGE gel; lane 5, zymogram analysis of KG51

547

enzyme on a 0.5% beechwood xylan-complemented PAGE gel. 25



Page 26 of 36

548

Figure 5. Enzymatic profiles of recombinant KG51 bifunctional enzyme at different temperature

549

and pH values. (A) Optimum pH for KG51 enzyme. Cellulase/xylanase activities were measured

550

using carboxymethyl cellulose (CMC, dotted lines) and beechwood xylan (solid lines) as

551

substrates at 40 and 50°C, respectively. Activities were assayed at pH range of 4–10: pH 4–6 (●)

552

in 50 mM sodium acetate buffer, pH 6–8 (○) in 50 mM sodium phosphate buffer, pH 8–10 (▼)

553

in 50 mM Tris/HCl buffer. (B) Optimum temperature of KG51 enzyme. Activities were assayed

554

at temperature range of 20–70°C using CMC (dotted lines) and beechwood xylan (solid lines) as

555

substrates, respectively at pH 5 in 50 mM sodium acetate buffer. (C) pH stability of KG51

556

enzyme, which was incubated at indicated pH at 4°C for 24 h. (D) Thermal stability of KG51

557

enzyme treated at indicated temperatures in 50 mM sodium acetate buffer (pH 5.0) for 1 h. Its

558

cellulase (dotted lines) and xylanase (solid lines) activities were measured. Highest activities in

559

all tests were defined as 100%. Experiments performed in triplicates were repeated

560

independently three times.

26


Page 27 of 36


Table 1. Comparison of amino acid sequence identifies between KG51 enzyme and other homologous proteins

Description

Identity (%)

GenBank accession number

Hypothetical protein [Eubacterium siraeum]

41

WP_005357523.1

Endoglucanase [Eubacterium siraeum V10Sc8a]

41

CBL34359.1

Endoglucanase [Ruminococcus sp. CAG:353]

39

CDE80894.1

Glycoside hydrolase family 5 [Pseudobacteroides cellulosolvens ATCC 35603 = DSM 2933]

38

KNY25463.1

Glycoside hydrolase [Pseudobacteroides cellulosolvens]

38

WP_036943060.1

Deduced amino acid sequence of novel KG51 gene was used for BLAST P searches using a query coverage cut-off of 97% and identity cut-off of 38%.

27



Page 28 of 36

Table 2. Substrate specificity of novel bifunctional KG51 enzyme toward various substrates Substrates

Main linkage types

Specific activity (U/mg)a

Carboxylmethyl cellulose (CMC)

β-1,4-glucan

3.79 ± 0.09

β-Mannan

β-1,4-mannan

3.28 ± 0.07

Beechwood xylan

β-1,4-xylan

2.63 ± 0.34

Laminarin

β-1,3/1,6-glucan

1.45 ± 0.04

Avicel

β-1,4-glucan

1.26 ± 0.01

Barley glucan

β-1,3/1,4-glucan

1.13 ± 0.03

Larchwood xylan

β-1,4-xylan

0.67 ± 0.11

P-Nitrophenyl-D-xylopyanoside

β-1,4-xylan

NDb

P-Nitrophenyl-D-glucopyranoside

β-1,4-glucan

ND

P-Nitrophenyl-D-cellobiose

β-1,4-glucan

ND

a

One unit (U) of enzyme activity corresponds to amount of enzyme releasing 1 µmol of reducing sugar in 1 min. Specific activity was calculated using 1% (w/v) substrates at optimum reaction conditions. All values correspond to means of triplicate independent experiments. b ND, activity not detected 561 562

28


Page 29 of 36


Table 3. Degree of polymerization (DP) profiles of 0.1% KGM and 0.1% KGMHs prepared with the KG51 enzyme analyzed by HPLC All saccharides of DP 1–6 (mg/mL)

Oligosaccharides of DP 2–6 (mg/mL)

Monosaccharide (mg/mL)

0.1% KGM

0

0

0

0.1% KGMHs

0.891±0.002

0.820±0.002

0.071±0.003

29



Page 30 of 36

Table 4. Population changes of Lactobacillus acidophilus KCTC 2182 grown in De Man, Rogosa, and Sharpe (MRS) broth only, MRS broth containing 0.1% konjac glucomannan (KGM) or 0.1% konjac glucomannan hydrolysates (KGMHs) Bacterial cell number (log cfu/mL) 0h

48 h

Increased cell numbers after 48 h (log cfu/mL)

MRS (Control)

3.92 ± 0.13

7.72 ± 0.11

3.79 ± 0.06a

MRS+0.1% KGM

3.92 ± 0.13

6.89 ± 0.06

2.97 ± 0.02b

Broth media

MRS+0.1% KGMHs 3.68 ± 0.01 8.07 ± 0.05 Different letters in a column indicate statistical differences at p < 0.05.

4.39 ± 0.05c

30


Page 31 of 36


Figures

Figure 1

31



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Figure 2

32


Page 33 of 36


Figure 3

33



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Figure 4

34


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Figure 5 563

35



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TOC graphic

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Metagenomic Mining and Functional Characterization of a Novel

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