Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen

Objective This study aimed to identify and characterize a novel endo-β-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. Methods A novel endo-β-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley β-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. Results The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40°C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley β-glucan (109.59±3.61 μmol/mg min) and Icelandic moss lichenan (35.35±1.55 μmol/mg min). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. Conclusion The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.


INTRODUCTION
Nonstarch polysaccharides (NSPs) in the plant cell wall, mainly cellulose and hemicellulose, make it challenging for animals to digest and absorb nutrients from plant-derived feedstocks.The presence of hemicellulose, one of the glucan-like NSPs in grain, acts as an anti-nutritional factor for monogastric animals, broiler chickens and pigs, resulting in increased chyme viscosity, and decreased feed utilization and growth performance [1,2].Strategies for eliminating the anti-nutritional effects of glucan have been proposed, using biophysical and biochemical approaches [3], of which enzymic digestion has become the favored method to degrade glucan-like NSPs, thereby reducing their undesirable viscosity.Glucanases are a group of glycoside hydrolases (GH) responsible for breakdown of glucanlike substrates into mono-and oligo-saccharides.In the carbohydrate-active enzyme (CAZy) database (https://www.cazy.org/),glucanases are categorized into endo-acting enzymes (endo-β-1,4-glucanase, EC 3.2.1.4;endo-β-1,3 [4] [4].Endo-acting glucanases degrade their substrates by randomly cleaving internal glycosidic bonds within the main polysaccharide chain [5].The anti-nutritional factor of undigestible glucan-like substrates is removed by glucanase digestion.More importantly, the polysaccharides are depolymerized into functional oligosaccharides, which are prebiotics for intestinal probiotic organisms, such as Lactobacillus sp. and Bifidobacterium sp.[6].Therefore, the exploration and characterization of endo-β-1,4-glucanases (EC 3.2.1.4)has great importance for the development of feed and food additives.
The herbivore rumen microbiome has garnered significant research interest due to its efficient digestion of cellulosic materials and its potential as a source of valuable CAZymes for industrial applications [12].Exogenous enzyme preparations are vital in poultry and swine production, improving nutrient utilization efficiency, mitigating the effects of unwanted components, expanding feed ingredient options, and enhancing formulation accuracy.They also contribute to environmental sustainability by reducing excreta moisture content, addressing various issues, and promoting intestinal health and immunity.Glucanases in these preparations hydrolyze polysaccharides into oligosaccharides, acting as prebiotics for intestinal microorganisms.Extracts from rumen microorganisms offer a valuable resource for discovering novel glucanases.This study focuses on leveraging these resources by expressing novel glucanase genes, exploring their enzymatic properties, deepening our understanding of these enzymes, and laying the foundation for the development of feed enzyme preparations.In this study, a novel GH9 glucanase gene, IDSGLUC9-4, was identified from sheep rumen microbes using previously obtained transcriptomic data [13].The gene was expressed heterologously in Escherichia coli, and the substrate specificity and product profile of the recombinant enzyme were characterized using a variety of glucan-like substrates.

Sequence analysis
Isoelectric point and molecular weight (pI, Mw) were predicted using the Expasy online tool (https://web.expasy.org/compute_pi/).Signal peptide and functional domain prediction were performed using InterPro (https://www.ebi.ac.uk/ interpro/).SWISS-MODEL was employed for comparative modeling of tertiary structures (https://swissmodel.expasy.org/interactive/).Multiple sequence alignment and phylogenetic analysis were conducted using MEGA software (v.11.0;Kumer Lab, Temple University, Philadelphia, PA, USA).Phylogenetic analysis used the Maximum Likelihood statistical method, employing the WAG correction model.The phylogeny assessment used the bootstrap method with 500 bootstrap replications.MEGA and SWISS-MODEL were used for this analysis.

Protein expression and purification
The transformation of the plasmid pET30a-IDSGLUC9-4 was carried out by heat shocking it into E. coli BL21(DE3) competent cells and streaking the mixture onto Luria-Bertani (LB) plates (5 g/L yeast extract, 10 g/L tryptone, 10 g/L sodium chloride, 18 g/L agar, 100 μg/mL kanamycin).Colonies containing the IDSGLUC9-4 gene were confirmed through colony-PCR and plasmid sequencing, and designated as BL21/pET30a-IDSGLUC9-4.The recombinant strain was added to LB medium (500 mL) with a 1% inoculum, then cultured at 37°C with continuously shaking at 180 rpm until the OD 600 reached 0.6 to 1.0.Then, IPTG (250 μL, 1 mol/L) was added to induce protein expression at 16°C with gentle agitation at 100 rpm for 16 h.The cultured cells were harvested by centrifugation at 4°C and 6,000×g for 10 min, then resuspended in 1× phosphate-buffered saline (50 mL).A 15-min sonication at 65% power, with a 4 s pause every 2 s, was performed using an ultrasonic disruptor (TZL-1200; Perwell, Suzhou, China) to yield the crude enzyme.The crude enzyme solution was treated with Affinity Ni-NTA agarose resin at a ratio of 1:50, then supplemented with 1 mL of 1 mol/L imidazole.Following this, the mixture was gently agitated at 100 rpm for 1 hour at 0°C.The mixture was gently loaded into a chromatography column and the target protein was eluted stepwise with phosphate buffer, containing 20, 50, or 250 mmol/L imidazole.The purified protein was used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and analysis of substrate selectivity and product profile.The enzyme substrate selectivity was tested on agar plates containing 0.2% (w/v) barley β-glucan, Icelandic moss lichenan, or konjac gum.Approximately 20 U of enzyme activity was spotted onto the plates, which were incubated for 16 h at 25°C, stained with a 0.1% (w/v) Congo red solution for 20 min, then destained with 1 mol/L NaCl until the clear areas became visible.

Enzyme characterization
Enzyme activity was assessed by the 3,5-dinitrosalicylic acid (DNS) reducing sugar assay [15], and the protein concentration was determined using the Bradford assay [16].One unit (U) of glucanase was defined as the amount of enzyme needed to generate 1 μmol of reducing sugar per min.Briefly, enzyme solution (40 μL, ~0.32 μg) and substrate (40 μL, 5 mg/mL of barley β-glucan, Icelandic moss lichenan, locust bean gum, or konjac glucomannan), were mixed and reacted for 15 min under the optimum conditions (40°C, pH 6.0), then the amount of reducing sugar produced was quantified with DNS; all assays were performed in triplicate.
Optimum temperature determination: Enzyme solution (40 μL, ~0.32 μg) was mixed with barley β-glucan solution (40 μL, 0.5% w/v) and the reaction was carried out at different temperatures (20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C) for 15 min, then DNS solution (80 μL) was added and incubated at 95°C for 10 min.After cooling to room temperature, the OD540 was determined using a microplate reader.In the control assay, deactivated enzyme solution (40 μL) was used, all other conditions remaining constant.The temperature corresponding to the maximum observed activity was designated as 100% and the relative activity at different temperatures was calculated.
Effects of metal ions and organic reagents on glucosidase activity: Recombinant glucosidase solution was mixed with solutions of different concentrations of various metal chlorides (KCl, NaCl, CaCl 2 , MgCl 2 , ZnCl 2 , CuCl 2 , MnCl 2 , NiCl 2 ), or mixed with different concentrations of organic solvents (ethylene diamine tetraacetic acid [EDTA], SDS, methanol, ethanol, propanol, butanol, dimethyl sulfoxide [DMSO]).After gentle centrifugation and mixing, the mixture was placed on ice for 30 minutes (metal chloride solution concentrations set at 1 mM or 5 mM, organic solvent concentrations set at 10 mM or 20 mM).The residual enzyme activity was determined under the influence of different metal ions and organic solvents, and calculated by comparison to the enzyme solution with only buffer added, which was considered 100%.
A more in-depth analysis of the hydrolysis products was performed by high-performance liquid chromatography (HPLC), with an LC-1200 instrument (Agilent Technologies, Santa Clara, CA, USA), fitted with an Asahipak NH2P-50 4E chromatography column (Shodex, Tokyo, Japan) and an RID-20A refractive index detector.The mobile phase was 65% aqueous acetonitrile, with a column temperature of 40°C and a flow rate of 1.0 mL/min.In addition, hydrolysis samples taken at 3 and 48 h were analyzed by Ultraflextreme MALDI-TOF/TOF (Bruker, Billerica, MA, USA).

Statistical analysis
The experiments conducted in this study, including protein concentration determination, DNS colorimetric reaction in enzymatic property studies, and quantification of oligosaccharide concentration in HPLC experiments, were all performed in triplicate.Image preparation and data analysis, including standard deviation and mean calculation, were conducted using GraphPad Prism v.8.0 (San Diego, CA, USA).Data were presented as the mean±standard deviation (n = 3).The section on "Effects of Metal Ions and Organic Reagents on Glucosidase Activity" involved single-factor comparisons.Statistical significance was analyzed using the Student's t-test integrated into GraphPad Prism (* p< 0.05; ** p<0.01; *** p<0.001).Significant differences were assessed by comparing each experimental group with the control group treated without inhibitors, with the control group set as 100%.

Glucanase gene mining and heterologous expression
Ruminants, such as sheep and cattle, are unable to digest cellulose and other plant cell wall polysaccharides, so they obtain most of their nutrition indirectly from the action of cellulolytic microorganisms in their rumens [18].Therefore, the rumen microbiome is a rich source of industrial CAZymes with high activity and useful properties.In this study, a GH9 β-glucanase gene, IDSGLUC9-4 (GenBank accession no.WUV41754.1),was isolated from sheep rumen microbes, using transcriptomic data obtained previously [13].The IDSGLUC9-4's open reading frame spanned 1,716 bp, encoding 571 amino acids with a theoretical molecular mass of 63.6 kDa and an isoelectric point of 6.12.Multiple sequence alignment suggested that the IDSGLUC9-4 gene shared high homology (>99%) with two other nucleotide sequences which annotated as GH family 9 protein and cellulase (GenBank accession no.MBR6110719.1 and MBE6326145.1),located in a metagenome-assembled genome annotated as Paludibacteracea bacterium and Bacteroidales bacterium.However, neither of these homologous enzymes has been functionally characterized.Phylogenetic analysis revealed that IDSGLUC9-4 and another enzyme (GenBank accession no.MBR6110719.1) from the Paludibacteraceae are both in the GH9 family (Figure 1).The catalytic mechanism of GH9 enzymes involves an inversion of anomeric stereochemistry.Cel9A, a processive endoglucanase from Thermobifida fusca, is active against bacterial cellulose and is the only known cellulase capable of independently degrading crystalline regions in bacterial cellulose, although it prefers amorphous regions [19].A closely related cellulase from Clostridium phytofermentans is the only GH9 cellulase encoded in its genome and is essential for cellulose degradation by the organism.Notably, this is the only documented instance of a single cellulase being essential for growth on cellulose [20].Detailed sequence alignment suggests that IDSGLUC9-4 and six other GH9 enzymes have five shared amino acid residues, three catalytic residues (Asp182, Asp184, and Glu560) and two conserved aromatic residues (Trp436 and Tyr545) (Figure 2) [21][22][23][24].
To investigate the biochemical properties of IDSGLUC9-4, the gene was expressed heterologously in E. coli to produce the recombinant enzyme.Following 6× His-tagged affinity purification, a prominent band at ~69 kDa was observed by SDS-PAGE (Figure 3A), consistent with its theoretical molecular weight of 63.4 kDa plus the expression vector backbone sequence of 5.3 kDa.Zymogram analysis indicated that IDSGLUC9-4 was active towards barley β-glucan, Icelandic moss lichenan, xyloglucan, and konjac gum (Figure 3B to 3E).
The GH9 family protein derived from the rumen of Hu sheep exhibits the capability to hydrolyze mixed-linkage glucans.Comparative analysis with previously characterized GH9 family glucanases reveals the presence of similar catalytic residues, indicating the successful isolation of a novel, uncharacterized glucanase from the sheep rumen.This enzyme represents a common exogenous enzyme preparation in livestock production.In-depth enzymatic and hydrolytic property studies will facilitate a comprehensive understanding of its functionality.

Biochemical properties of recombinant IDSGLUC9-4
The optimum pH of IDSGLUC9-4 was 6.0, with >75% of the maximum catalytic activity between pH 5.0 to 7.0 (Figure 4A).IDSGLUC9-4 was relatively stable (> 70%) between pH 4.0 to 6.0 (Figure 4B) and most stable at pH 6.0, but relatively unstable above pH 7.0.Notably, after preincubation for 1 h at pH 4.0 and 5.0, the residual activities were 78.22% and 89.53%, respectively, indicating that the enzyme and its unknown producing microorganism were well-adapted to the acidic environment of the rumen [17,18].The optimum temperature for IDSGLUC9-4 was 40°C (Figure 4C) and the enzyme was much less stable at higher temperatures.Thermostability assays revealed that the IDSGLUC9-4 was sensitive to heat-challenge; after 1 h preincubation at 30°C and 40°C, the enzyme retained 75.10%±0.43%and 56.33%±1.14% of its initial activity, respectively (Figure 4D), and above 50°C, the activity decreased rapidly.These findings suggested that IDSGLUC9-4 was a relatively acid-resistant and mesophilic enzyme, which is consistent with the properties of other gastrointestinal tract-derived CAZymes reported previously [25][26][27][28].
Substrate selectivity analysis revealed that IDSGLUC9-4 could hydrolyze barley β-glucan, Icelandic moss lichenan, konjac glucomannan and tamarind xyloglucan; barley β-glucan was the substrate with the highest catalytic activity, 109.59±3.61 μmol/mg min (Table 1).IDSGLUC9-4 was inactive towards beechwood xylan, galactomannan, guar gum, arabinan, Laminaria digitata laminaran, locust bean gum and arabinoxylan.The effects of metal ions and organic compounds on the activity of the enzyme, with β-glucan as substrate, were also determined at 40°C (Table 2).All the metal ions inhibited the enzyme, with Mn 2+ the strongest in-hibitor, as did all the organic compounds, with propanol and methanol the strongest inhibitors.Previously reported β-glucanases have diverse behaviors under the influence of metal ions and organic compounds.The activity of an exo-β-1,3glucanase from the moose rumen microbiome [29] more than doubled in the presence of zinc ions, retaining normal activity in the presence of EDTA, propanol, and butanol.In contrast, the activity of IDSGLUC9-4 slightly decreased in the presence of 20 mM EDTA (p<0.05) but lost >50% of its activity in the presence of DMSO and propanol.
Enzymatic property studies revealed that this novel glucanase exhibits a specific activity of 109.59±3.61μmol/mg min towards β-glucan.It shows stability within the temperature range of 30°C to 40°C.Additionally, it demonstrates tolerance to acidity, maintaining over 75% activity after 1 hour of incubation at pH 4.0 to 5.0.These preferences for moderate temperature and tolerance to weak acidity align with its origin from the rumen environment.Moreover, its activity towards four different polysaccharide substrates suggests promising Figure 1.Phylogenetic analysis of IDSGLUC9-4 (GenBank accession no.WUV41754.1).Multiple sequence alignment was performed and used to generate a phylogenetic tree using MEGA11.0 and the maximum likelihood (ML) statistical method based on the WAG correction model, with a bootstrap of 500.The tree was drawn to scale (length = 0.1), and branch length was measured according to the number of substitutions per site.application potential for IDSGLUC9-4.Further investigation into its hydrolytic mechanism is required to comprehensively elucidate its enzymatic properties.
To further analyze the product profiles of IDSGLUC9-4, mono-and oligosaccharides were used as hydrolysis substrates (Figures 5D, 7D).TLC (Figure 5D) clearly showed that both G5 and G4 were effectively hydrolyzed by IDS-GLUC9-4, generating G2 and G3 from G5 and G2 as the final product from G4. Quantitative determination by HPLC (Figures 7) showed that G5 was initially cleaved into equivalent amounts of G3 and G2, then the G3 concentration began to decrease at 1 h, whereas the G2 concentration increased, indicating hydrolysis of G3 into G2 (Figures 7A, 7A').After 48 h, the products derived from G4 were G2 (2,007.05±31.11nmol/L) and G3 (151.40±5.13nmol/L), whereas those from G5 were G2 (1,425.18±49.28nmol/L) and G3 (461.40 ±40.38 nmol/L).IDSGLUC exclusively cleaved G4 into two molecules of G2 (Figures 7B, 7B'), but G2 was highly resistant to further hydrolysis; its concentration decreased only slightly from that at 3 h, even after 48 h (Figures 7D, 7D').Hydrolysis of G3 into G2 (Figure 7C, 7C') was incomplete and glucose was not detected, indicating that the glycosylation activity of the enzyme partially reversed the G3 hydrolysis and consumed all the glucose produced [9].Notably, unlike the complete degradation of G4 and G5, the residual G3 concentration after 48 h was 304±15.27nmol/L (29.3% of the initial amount), suggesting that IDSGlUC9-4 preferentially cleaves high-DP cello-oligosaccharides.In addition, glucose as substrate decreased from 3.070±0.085to 0.866±0.014(28.2%   of the initial amount) after 48 h (Figure 7E and 7E'), indicating the formation of oligosaccharides and consistent with the glycosylation activity of IDSGlUC9-4.
The analysis of the degradation mechanism of polysaccharides and oligosaccharides by IDSGLUC9-4 reveals its typical endo-glucanase activity, capable of hydrolyzing polysaccharide substrates containing β-1,4-glycosidic bonds to produce oligosaccharides with DP>2, with predominant hy-drolysis products being G2, G3, and G4.Its action on fibrous oligosaccharides G1-G5 demonstrates the ability to hydrolyze oligosaccharides with DP≥3 into G2 and G3.The hydrolytic potential of polysaccharides and oligosaccharides suggests suitability as a feed enzyme preparation.It can hydrolyze cellulose, which is difficult for monogastric animals to digest, into oligosaccharides that are readily absorbable or utilizable by intestinal probiotics.The substrate spectrum of the enzyme includes β-glucan, lichenin, xylan, and laminarin, indicating its ability to degrade not only β-glucans with mixed linkages of 1,3-1,4 but also polysaccharides with other linkage forms.Future efforts may focus on enhancing the activity of recombinant glucanases through molecular evolution engineering, for application in the development of enzyme preparations for agricultural waste treatment or animal feed.

CONCLUSION
The study identified a novel, uncharacterized GH9 family protein, IDSGLUC9-4, originating from the rumen of Hu sheep.Recombinant glucanase was heterologously expressed and functionally characterized, revealing it as an endo-glucanase with relative acid tolerance and preference for moderate temperatures.It exhibits activity towards various substrates including β-glucan, lichenan, xyloglucan, and konjac gum, completely hydrolyzing polysaccharides into oligosaccharides of G2, G3, and G4.This glucanase holds promising prospects as a feed enzyme preparation for monogastric animals, particularly in the hydrolysis and release of prebiotic oligosaccharides.Future investigations could explore enhancements through molecular evolution engineering, immobilization, and other methods to broaden its application potential in livestock production.