Mannooligosaccharides (M6, mannohexaose; M5, mannopentaose; M4, mannotetraose; M3, mannotriose; and M2, mannobiose) were obtained from Megazyme (Wicklow, Ireland). Locust bean gum (LBG) and β-xylanase (XYL) from Thermomyces lanuginosus and commercial cellulase (CEL) from Trichoderma reesei (ATCC 26921) were obtained from Sigma-Aldrich (USA).

The complete β-mannanase gene (pman) was discovered in the genome sequence of T. asperellum ND-1. By utilizing SignalP-5.0 software (http://www.cbs.dtu.dk/services/SignalP/), we predicted that TaMan5 possesses a 27-residue signal peptide (MGNRALNSMKFFKSQALALLAATSAVA). To optimize the full length for yeast codon preference (http://www.jcat.de/), pman gene was synthesized and then ligated to the pPICZαA vector (forming pPICZαp-oman) by Tsingke Corp. (Beijing).

Enzyme activity of TaMan5 was further increased by replacing α-factor with propeptide in P. pastoris. The recombinant plasmids, pPICZp-oman (with a propeptide sequence) and pPICZα-oman (with an α-factor signal peptide), were cloned from the synthesized vector pPICZαp-oman with the corresponding primer pairs (pPICZp-oman-F/pPICZp-oman-R and pPICZα-oman-F/pPICZα-oman-R) (Supplementary Table S1). The expression vectors, pPICZp-oman and pPICZα-oman, were linearized by SacI, introduced into P. pastoris through electroporation, and screened based on the manufacturer’s protocol (Invitrogen, San Diego, CA). Transformants were grown on agar plates containing peptone dextrose (YPD) medium supplemented with 100 μg/mL Zeocin (Invitrogen, Carlsbad, CA, United States) to obtain one gene copy. The primer pair AOX-F/AOX-R was used to confirm the positive strains by PCR. The constructed strains were designated as α-oTaMan5 and p-oTaMan5.

To produce β-mannanase TaMan5, recombinant strains of α-oTaMan5, p-oTaMan5, and X-33 were incubated in BMMY medium as previously reported (Zheng et al., 2018). All strains were grown at 30°C, with shaking at 200 rpm for a duration of six days. Throughout the fermentation period, the methanol concentration was maintained at 1.0 by supplementing with 100% (v/v) methanol (Basit et al., 2018). The fermentation broth was subjected to centrifugation (4°C, 5,000 rpm) to collect crude enzyme. The TaMan5 expression was analyzed in 12% SDS-PAGE, and its concentration was analyzed based on the Bradford method (Bradford, 1976). All media and operation steps were executed following the guidelines provided in the P. pastoris expression manual (Invitrogen, San Diego, CA).

TaMan5 activity was tested using 3,5-dinitrosalicylic acid (DNS) (Miller, 1959) at a temperature of 65°C, with LBG serving as the substrate. The reaction mixture consisted of 0.45 mL sodium acetate buffer (pH 4.0) containing 0.05 mL appropriate diluted enzyme and 0.5% (w/v) LBG. The reaction was terminated by adding 0.75 mL DNS and boiling the mixture for 5 min. The absorbance of the solution was assayed at 540 nm, with one unit (U) activity regarded as the amount of enzyme capable of liberating 1 μmol of reducing sugars per minute.

The impact of various concentrations of NaCl and ethanol on the activity of TaMan5 was evaluated. TaMan5 was subjected to a 1-h incubation at 4°C in 50 mM sodium acetate solution (pH 4.0) with varying final concentrations of NaCl or ethanol, ranging from 0% to 20% (w/v). After incubation, residual enzymatic activity was separately assayed and compared with the control (without any additives). All assays were analyzed in triplicate.

Transglycosylation of TaMan5 toward mannotriose/mannotetraose was carried out by incubating 50 mg/mL mannotriose/mannotetraose with 1 U of TaMan5. At various time points, hydrolytic products (25 μL) were collected, boiled for 10 min, and then analyzed using thin-layer chromatography (TLC) following the method described by Katrolia et al. (2013). Substrates containing inactive enzymes were used as blank controls.

For TLC analysis, silica gel plates (Merck Silica Gel 60 F₂₅₄, Germany) were used for separation, with a mobile phase consisting of n-propanol/ethanol/water (7:1:2, v/v/v). After development, the saccharides on the plates were visualized by immersing them in a solution of 5% sulfuric acid in methanol, followed by heating at 100°C for 15 min. Mannooligosaccharides (DP 2–6) and mannose were employed as the standard for the comparison and identification of the products.

Lignocellulosic biomass (bagasse and corn stover) was pretreated by using an alkaline method as previously reported (Basit et al., 2018). The enzymatic degradation of 5% (w/v) corn stover or bagasse by CEL (15 U/g biomass), XYL (47 U/g biomass), and TaMan5 (55 U/g biomass) individually or in combination was performed at 50°C, with constant mixing for 72 h at 200 rpm. Blank controls containing only the substrate were also performed. After centrifugation for 5 min at 12,000 rpm, the supernatants were obtained, and the quantification of reducing sugars released from the biomass was assayed using the DNS method (Miller, 1959).

Several strategies have been employed to achieve optimal expression levels of heterologous proteins in P. pastoris (Looser et al., 2015). Utilizing the secretion pathway of P. pastoris, which limits endogenous protein secretion, is considered an effective and desirable approach for recombinant protein production (Šiekštelė et al., 2015). The selection of appropriate signal peptide sequences has been recognized as a crucial factor in enhancing protein expression in P. pastoris, focusing on improving the secretion level and recruitment of different signal peptides (Vadhana et al., 2013; Zheng et al., 2018; Püllmann and Weissenborn, 2021).

To enhance the enzyme activity of TaMan5, the α-factor was substituted with a propeptide. Two different recombinant vectors, pPICZp-oman (containing the propeptide sequence) and pPICZα-oman (containing the α-factor signal peptide), were designed. The recombinant vectors were transformed into P. pastoris, and engineering strains, α-oTaMan5 and p-oTaMan5 (Figure 1), were selected on YPD medium supplemented with Zeocin (100 μg/mL).

Following induction with 1% (v/v) methanol for 6 days, TaMan5 activity of the recombinant strain p-oTaMan5 (with the propeptide sequence) continuously increased, reaching a maximum of 93.56 ± 2.17 U/mL (779.67 ± 18.08 U/mg) (Figure 2A), which was remarkably 1.35-fold higher than that of α-oTaMan5 (with the α-factor signal peptide). Previous studies have demonstrated that the recruitment of natural propeptides, such as trypsin from Streptomyces griseus (Zhang et al., 2018) and α-L-arabinofuranosidase from Aspergillus niger ND-1 (Zheng et al., 2018), can remarkably enhance the production levels of heterologous proteins in P. pastoris. SDS-PAGE results revealed that the recombinant proteins secreted by p-oTaMan5 have the molecular weight of approximately 66 kDa (Figure 2B). Protein expression in yeast is transferred through a secretory system from the endoplasmic reticulum to Golgi bodies and finally to the extracellular space (Nett et al., 2011). Taken together, these findings suggested that the recruitment of a propeptide is an effective and potential strategy for improving TaMan5 activity.

The impact of varying concentrations of ethanol and NaCl on TaMan5 activity was evaluated (Figure 3). TaMan5 retained nearly full activity across a wide range of ethanol concentrations (0%–20%) (Figure 3A), whereas rManAJB13 from Sphingomonas sp. JB13 retained 69.1% of the β-mannanase activity at 10% of ethanol concentration (Zhou et al., 2012). Ethanol-tolerant β-mannanases could potentially be applied in bioethanol production, and TaMan5 has potential application in the process of biocatalysts. Similar to other hemicellulases, including β-mannanases (Jiang et al., 2006) and β-xylanases (Yegin, 2017), TaMan5 exhibited higher activity at 15% of salt concentration (Figure 3B), suggesting that it might be a very beneficial additive in the processing of seafoods and saline foods, such as sauces and pickles.

Hydrolysis properties of TaMan5 showed that it required at least three mannose (mannotriose) residues as the substrate for the production of mannobiose as described previously (Zheng et al., 2023). Mannotriose was completely hydrolyzed into mannobiose, and no mannose was detected, indicating that TaMan5 exhibited transglycosylation as well as hydrolysis function. Moreover, when incubated with a high concentration of mannotriose (Figure 4A) or mannotetraose (Figure 4B), the transglycosylation action of TaMan5 can be detected, yielding the corresponding production of mannotetraose or MOS (DP > 4) (Figure 4). Interestingly, Asp¹⁵² is positioned opposite to the enzyme’s active region (Glu³¹³ and Glu²⁰⁵) based on the 3D structure of TaMan5 (Figure 5A). Thus, we deduced that Asp¹⁵² is probably responsible for the binding of substrates to catalytic residues. Moreover, the distance between crucial catalytic sites was determined using PyMOL software, and the inter-residue distances of Glu²⁰⁵–Glu³¹³ and Glu³¹³–Asp³⁵⁷ are ∼4.5 and 7.8 Å, respectively (Figure 5K), which was much shorter than those of Glu²⁰⁵–Asp¹⁵² (11.2 Å) and Glu³¹³-Asp¹⁵² (12.0 Å) (Figure 5K). Based on 3D structure analyses, mutant activity, and space distance, Asp¹⁵² can promote the combination of mannotriose/mannotetraose and active center, the catalytic sites (Glu³¹³–Asp³⁵⁷) are responsible for transglycosylation action, and Glu²⁰⁵–Glu³¹³ are crucial sites for hydrolysis function.

To better elucidate the action mode of TaMan5, molecular docking analysis was carried out (Figure 5). Mannotriose, mannotetraose, and mannan were docked into the catalytic region comprising Glu²⁰⁵, Glu³¹³, Asp¹⁵², and Asp³⁵⁷ residues (Figures 5A–J). For mannotetraose, catalytic residues Glu³¹³ and Glu²⁰⁵ of TaMan5 degrade mannotetraose into mannose and mannotriose (Figure 5B), and Glu³⁵⁷ and Glu³¹³ then promote the formation of an intermediate (mannopentose) (Figure 5B) from new substrates (mannose and mannotetraose) for further hydrolysis by catalytic sites (Glu³¹³ and Glu²⁰⁵) (Figure 5C), generating mannobiose and mannotriose (Figure 5C). For mannotriose, Glu³¹³ and Glu²⁰⁵ transform mannotriose into mannose and mannobiose (Figure 5F), and Glu³⁵⁷ and Glu³¹³ then promote the formation of intermediate (mannotetraose) (Figure 5F) from new substrates (mannose and mannotriose) for further degradation (Figure 5G), generating mannobiose (Figure 5H). The catalytic mechanism of TaMan5 involves the transglycosylation of mannose to mannotriose or mannotetraose as the substrate to produce a mannotetraose or mannopentose intermediate, respectively (Figure 5I). Based on hydrolysis and transglycosylation process of other β-mannanases reported previously (Jiang et al., 2006; Do et al., 2009; Katrolia et al., 2013), this study proposed a novel degradation process of mannotetraose: 2 M4 + E = 2 M4(E); 2 M4(E) = M3 + (M1 + M4)(E); (M1 + M4)(E) + M3 = M5(E) + M3; M5(E) + M3 = M2 + 2 M3 + E, where 2 M4 represents two mannotetraoses; E, free TaMan5; M4(E), TaMan5–mannotetraose complex; M3, mannotriose; (M1 + M4) (E), mannose–mannotetraose–TaMan5 complex; M1, mannose; M5(E), TaMan5–mannopentose complex; M5, mannopentose; M2, mannobiose; M3, mannotriose; and 2 M3, two mannotrioses. For mannotriose, 2 M3 + E = 2 M3(E); 2 M3(E) = (M1 + M3)(E) + M2; (M1 + M3)(E) + M2 = M4(E) + M2; M4(E) + M2 = 3 M2 + E (where M3(E) represents the TaMan5–mannotriose complex; (M1 + M3)(E), mannose–mannotriose–TaMan5 complex; 3 M2, three mannobioses; and other abbreviations as mentioned previously), which are consistent with TLC results, 3D structure, site-directed mutagenesis, and space distance of crucial catalytic sites Glu²⁰⁵, Glu³¹³, and Asp³⁵⁷ in the degradation of mannan to produce mannobiose. These results illustrate the diverse functionality of GH5 β-mannanases from fungi and offer valuable reference data to guide future advancements in enzyme engineering and biocatalysis.

In natural systems, the efficient breakdown of biomass polysaccharides relies on the cooperative action of diverse carbohydrate-active enzymes (CAZymes), particularly cellulases and hemicellulases (Gao et al., 2011; Bhattacharya et al., 2015; Mattam et al., 2022). As shown in Figure 6, the addition of CEL (15 U/g biomass) resulted in the degradation of approximately 21.5% and 27.4% of bagasse and corn stover, respectively, following a 72-h incubation at 50°C and pH 4.0. When xylanase and mannanase were supplemented alongside cellulases, a synergistic effect was observed, leading to significantly improved saccharification levels. Specifically, the saccharification levels increased to 43.5% for corn stover (Figure 6A) and 37.5% for bagasse (Figure 6B). This enhanced saccharification efficiency highlights the cooperative action of these enzymes and underscores their importance in biomass degradation processes.

Furthermore, the enzymatic degradation level, also referred to as the conversion of reducing sugars, was significantly enhanced when XYL and TaMan5 were added together and was 58.8% and 42.7% higher than that of CEL alone toward corn stover and bagasse (Figure 6). The removal of hemicellulose and the improved degradation efficiency can exert a cooperative action by enhancing the accessibility of cellulases to cellulose. This, in turn, allows for the reduction of enzyme dosages, representing a sustainable approach for the production of value-added products from agricultural waste (Jeon et al., 2011; Moraïs et al., 2011; Várnai et al., 2011; Rakotoarivonina et al., 2016).