Expression of the Thermobiﬁda fusca β -1,3-Glucanase in Yarrowia lipolytica and Its Application in Hydrolysis of β -1,3-Glucan from Four Kinds of Polyporaceae

: The gene encoding a thermostable β -1,3-glucanase was cloned from Thermobiﬁda fusca and expressed constitutively by Yarrowia lipolytica using plasmid pYLSC1. The expression level of the recombinant β -1,3-glucanase reached up to 270 U/mL in the culture medium. After a treatment with endo- β -N-acetyl-glucosaminidase H, the recombinant protein appeared as a single protein band, with a molecular size of approximately 66 kDa on the SDS-polyacrylamide gel. The molecular weight was consistent with the size predicted from the nucleotide sequence. The optimum temperature and pH of the transformant β -1,3-glucanase were 60 ◦ C and pH 8.0, respectively. This β -1,3-glucanase was tolerant to 10% methanol, ethanol, and DMSO, retaining 70% activity. The enzyme markedly hydrolyzed Wolﬁporia cocos and Pycnoporus sanguineus glucans. The DPPH and ABTS scavenging potential, reducing power and total phenolic contents of these two Polyporaceae hydrolysates, were signiﬁcantly increased after 18 h of the enzymatic reaction. The present results indicate that T. fusca β -1,3-glucanase from Y. lipolytica transformant (pYLSC1- 13g ) hydrolyzes W. cocos and P. sanguineus glucans and improves the antioxidant potential of the hydrolysates.


Introduction
Edible medicinal fungi have been used in China for over two thousand years, and relevant information is available in historical relics. Numerous Polyporaceae are often used in Chinese medicine and dietary conditioning, such as Wolfiporia cocos (Fu-Ling), Pycnoporus sanguineus (Red fungus), Polyporus umbellatus (Chu-Ling), Laccocephalum mylittae (Lei-Wan), since they are rich in β-1,3 glucan.
Studies on traditional medicine have reported that β-glucan has anti-inflammatory, anti-allergic, antitumor, immunoregulatory, and antioxidant activity; thus, preventing bacterial infections and regulating other physiological phenomena [1]. β-glucan is one of the principal components of fungal cell walls. These medicinal fungi are rich in β-glucan, but have different chemical compositions. Polysaccharides with β (1 → 3) and β (1 → 6) or β (1 → 4) and β (1 → 6) glycosidic bonds, rather than those with pure β (1 → 4) glycosidic bonds, usually have stronger pharmacological activity [2,3]. The previous reports show that the low molecular weight β-glucans of oat and yeast reveals anticancer activity, antitumor, and immunological properties, respectively [4,5]. Bioactivity of polysaccharides is highly related to their chain structure, chemical composition, molecular weight, backbone, and degree of branching [6,7]. β-glucans from various fungi vary greatly in length and the degree of branching. Although the flavonoids, saponins, tannins, and terpenes are antioxidant compounds in the mushroom [8]. Therefore, the antioxidant properties of fungi Processes 2021, 9, 56 2 of 11 are primarily attributed to β-glucan [9]. β-1,3 glucanase has been used to destroy cell walls of some fungi and crack the fragments the fragments having immunoregulatory activity [10]. It is able to reduce molecular weight polysaccharide for β-1,3-glucan to produce low molecular weight β-1,3-glucan with pharmacological activity properties [11]. It has been reported that, Ganoderma lucidum β-1,3 glucan with low molecular weight and low molecular weight β-glucan of yeast had better immunological and antioxidant activities, respectively [4,12].
W. cocos are used in approximately 10% of traditional Chinese medicine preparations. W. cocos contains abundant β-1,3-glucan and is the principal component. Insolubility of β-1,3-glucan has low biological activity and can be chemically modified to increase its hydrophilicity. W. cocos β-1,3-glucan has various bioactivities [13], and its low molecular weight facilitates free radical scavenging activity and helps to prevent DNA damage [14]. P. sanguineus β-1,3-glucan reportedly scavenges DPPH free radicals [15] and has antimicrobial and antitumor effects [16], and its ethanol extract has different biological activities. P. umbellatus is used to treat various symptoms including edema, oliguria, and jaundice [17]. Typical of other β-glucans, P. umbellatus polysaccharide (β-1,3-glucan) reportedly has anticancer, hepatoprotective, immunoregulatory, and antitumor activity, and prevents kidney damage [18]. P. umbellatus polysaccharide has received increasing attention owing to its prominent physiological and biological functions [19]. L. mylittae is a traditional Chinese medicinal fungus. It has been used as a vermifuge against many kinds of parasites, including roundworms, cestodes, and ancylostoma [20].
In order to reduce energy costs in the production process, a mesophilic host is often used as the host to express the enzymes originally isolated from thermophilic organisms. Some available mesophilic expression systems have been reported. E coli expression system is usually the first choice because of its simple genetic manipulation, rapid growth, and high transformation efficiency. The inclusion body formation and intracellular accumulation limit its use [24]. The Pichia pastoris expression system was another option for mesophilic expression. However, P. pastoris showed low transformation efficiency and high false positives in transformant. Yarrowia lipolytica, generally regarded as safe (GRAS) yeast, also serves as a mesophilic host for heterologous protein expression [25]. There are many optional gene engineering tools for use in this host-vector system [26].
Therefore, this study aimed to constitutively express heterologous the β-1,3-glucanase gene in a Y. lipolytica mesophilic expression system and hydrolyze polysaccharides from edible-medicinal Polyporaceae, and to evaluate the antioxidant properties of their partial hydrolysates.

Materials and Microorganisms
Whole, dry sporocarps of P. sanguineus, W. cocos, P. umbellatus, and L. mylittae were purchased from a local Chinese pharmacy in Taichung, Taiwan, in February 2020. They were identified and stored by the Department of Cosmetic Science, Providence University, Taiwan. The actinomycetes, Thermobifida fusca BCRC 19214, isolated from a compost sample collected in Taiwan and stocked in Bioresource Collection and Research Center (BCRC) in Hsinchu, Taiwan was used herein [22]. Yarrowia lipolytica expression system (strain P01g and pYLSC1) was purchased from Yeastern Biotech Co., Ltd. (Taipei, Taiwan) [24]. The VioTag DNA polymerase, PCR buffer, and dNTP were purchased from Viogene (Sunnyvale, CA, USA). The YPD (Yeast Extract Peptone Dextrose) medium (Y1500), yeast nitrogen base without amino acids (Y0626), agar, restriction endo endodeoxyribonucleases, T4 DNA ligation kit, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Construction of the β-1,3-Glucanase Expression Plasmid
The β-1,3-glucanase gene was amplified from the chromosomal DNA of T. fusca BCRC 19214 as template by PCR using the primers 5 -AAA GGC CGT TCT GGC CGT CCG ACT AGG CTC CGG C-3 (SfiI site is underlined) and 5 -AAA GGT ACC TCA ATG ATG ATG ATG ATG ATG CCC GGT CGC CAA CTG C-3 (KpnI site is underlined) based on the gene sequence (NCBI accession number WP_011292553.1) of T. fusca YX. The amplified PCR product was further digested with SfiI and KpnI, and then ligated with SfiI-KpnItreated pYLSC1. The resulting plasmid pYLSC1-13g ( Figure 1) was used for recombinant enzyme production in Y. lipolytica P01g. DNA manipulation was carried out as previously reported [27].

Construction of the β-1,3-Glucanase Expression Plasmid
The β-1,3-glucanase gene was amplified from the chromosomal DNA of T. fusca BCRC 19214 as template by PCR using the primers 5′ -AAA GGC CGT TCT GGC CGT CCG ACT AGG CTC CGG C-3′ (SfiI site is underlined) and 5′-AAA GGT ACC TCA ATG ATG ATG ATG ATG ATG CCC GGT CGC CAA CTG C-3′ (KpnI site is underlined) based on the gene sequence (NCBI accession number WP_011292553.1) of T. fusca YX. The amplified PCR product was further digested with SfiI and KpnI, and then ligated with SfiI-KpnI-treated pYLSC1. The resulting plasmid pYLSC1-13g ( Figure 1) was used for recombinant enzyme production in Y. lipolytica P01g. DNA manipulation was carried out as previously reported [27].

Transformation and Screening of Y. lipolytica Transformant
The NotI-linearized pYLSC1-13g was introduced into Y. lipolytica P01g [25]. The transformants cells were spread on agar plates, which contained 6.7 g/L yeast nitrogen base without amino acids (Y0626), 20.0 g/L glucose, and 15.0 g agar/L, and cultivated at 28 °C for 4 days. The selected transformants were grown in 50 mL YPD medium (Y1500) in 250-mL Hinton flask, 28 °C, 200 rpm, and those with apparent β-1,3-glucanase activity were chosen for further study. Integration of the target gene into chromosome of Y. lipolytica P01g was checked by genomic PCR.

Enzyme Expression and Purification
Y. lipolytica transformant (pYLSC1-13g) with high-β-1,3-glucanase-activity was incubated in 200 mL YPD medium in 500-mL Hinton flasks on a reciprocal shaker (200 rpm) at 28 °C for 120 h. After cultivation, the culture supernatant was collected by centrifugation at 10,000 ×g under 4 °C for 30 min and served as a crude enzyme solution.
The crude enzyme solution was applied to a 10 mL Ni 2+ -NTA column (Merck KGaA, Darmstadt, Germany). After washed with 100 mL wash buffer (50 mM phosphate (pH 7.5), and 10 mM imidazole), the protein bound to the column was eluted with elution buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl and 500 mM imidazole). The eluted fractions containing the purified enzyme were collected.

Transformation and Screening of Y. lipolytica Transformant
The NotI-linearized pYLSC1-13g was introduced into Y. lipolytica P01g [25]. The transformants cells were spread on agar plates, which contained 6.7 g/L yeast nitrogen base without amino acids (Y0626), 20.0 g/L glucose, and 15.0 g agar/L, and cultivated at 28 • C for 4 days. The selected transformants were grown in 50 mL YPD medium (Y1500) in 250-mL Hinton flask, 28 • C, 200 rpm, and those with apparent β-1,3-glucanase activity were chosen for further study. Integration of the target gene into chromosome of Y. lipolytica P01g was checked by genomic PCR.

Enzyme Expression and Purification
Y. lipolytica transformant (pYLSC1-13g) with high-β-1,3-glucanase-activity was incubated in 200 mL YPD medium in 500-mL Hinton flasks on a reciprocal shaker (200 rpm) at 28 • C for 120 h. After cultivation, the culture supernatant was collected by centrifugation at 10,000× g under 4 • C for 30 min and served as a crude enzyme solution.
The crude enzyme solution was applied to a 10 mL Ni 2+ -NTA column (Merck KGaA, Darmstadt, Germany). After washed with 100 mL wash buffer (50 mM phosphate (pH 7.5), and 10 mM imidazole), the protein bound to the column was eluted with elution buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl and 500 mM imidazole). The eluted fractions containing the purified enzyme were collected.

Determination of β-1,3-Glucanase Activity
The 1 mL reaction solution was prepared by mixing 0.1 mL of the enzyme solution, 0.8 mL of Tris-HCl buffer (20 mM, pH 8.0) and 0.1 mL of laminarin (10 mg/mL), followed by incubation at 60 • C. The reducing sugars resulting from laminarin hydrolysis were assayed by the DNS assay [28]. One unit of β-1,3-glucanase activity was defined as the amount of enzyme that release 1 µg glucose per min at 60 • C. To determine the metal effect on the enzymatic activity, 1 mM metal ion, including Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , Cu 2+ , Mn 2+ , Co 2+ , and Hg 2+ was added into the reaction solution before incubation at Processes 2021, 9, 56 4 of 11 60 • C. Solvents including methanol, ethanol, isopropanol, DMSO, acetonitrile, and acetone was individually included in the reaction solution at 10% or 20% to determine the solvent stability of the enzyme.

Hydrolysis of Polyporaceae Glucans
Various Polyporaceae were placed in 100-mL serum bottles, and β-1,3-glucanase and 20 mM Tris-HCl buffer (pH 8.0) were added to adjust the total volume to 80 mL, followed by hydrolysis for 24 h at 60 • C. The hydrolysis degree was computed based on the following equation: Hydrolysis degree (%) = (The total sugar of the supernatant (µg)/substrate (g)) × 100%.

Quantification of Reducing Sugars and Total Sugar
Levels of reducing sugars were directly determined via the 2,4-dinitrosalicylic acid (DNS) assay. The total sugar contents of the samples were measured through the phenolsulfuric acid method.

Statistical Analysis
All data were analyzed in triplicate. The mean values were compared to the appropriate control using Student's t-test. p-values less than 0.05 indicated statistically significant differences.

Purification of β-1,3-Glucanase from Y. lipolytica Transformant (pYLSC1-13g)
β-1,3-glucanase in the culture supernatant was purified using a Ni 2+ -NTA column as described in the Materials and Methods Section (Section 2). The yield and purification fold of the process was 29.27% and 3.93-fold, respectively ( Table 1). The miscellaneous proteins in the culture supernatant of the Y. lipolytica transformant was fewer than that of the E. coli expression system. This will accelerate the application of the β-1,3-glucanase in the industrial process conveniently without numerous purification procedures.

Purification of β-1,3-Glucanase from Y. lipolytica Transformant (pYLSC1-13g)
β-1,3-glucanase in the culture supernatant was purified using a Ni 2+ -NTA column as described in the Materials and Methods Section (Section 2). The yield and purification fold of the process was 29.27% and 3.93-fold, respectively ( Table 1). The miscellaneous proteins in the culture supernatant of the Y. lipolytica transformant was fewer than that of the E. coli expression system. This will accelerate the application of the β-1,3-glucanase in the industrial process conveniently without numerous purification procedures. As shown in Figure 3, the purified β-1,3-glucanase from Y. lipolytica transformant (pYLSC1-13g) was obtained as a single protein band on SDS-PAGE, with an estimated molecular weight of approximately 66 kDa. The optimal pH and temperature of the purified β-1,3-glucanase were pH 8.0 and 60 • C, respectively, with approximately >85% activity retained at 40~60 • C for 4 h. Enzyme activity decreased rapidly at 70 • C, with only 10% activity retained after 1 h of incubation ( Figure 4).
After 18 h of hydrolysis, the hydrolysis degree for W. cocos and P. sanguineus were 40% and 6%, respectively, while those for P. umbellatus and L. mylittae were <1%.

Evaluation of the Antioxidant Activity of Polyporaceae Hydrolysates
After glucans from the aforementioned the W. cocos and P. sanguineus were hydrolyzed by β-1,3-glucanase for 18 h, the antioxidant activities of enzyme hydrolysates were determined. The free radical scavenging assay revealed a significant increase in the DPPH scavenging potential of the two hydrolysates ( Figure 8A).
The DPPH scavenging potential of P. sanguineus hydrolysates was consistent with an increase in its hydrolysis time. The maximum DPPH scavenging potential approached 2.3-fold after 18 h of hydrolysis. On hydrolyzing W. cocos glucans with β-1,3-glucanase for 1 h, the DPPH scavenging potential peaked and plateaued with an increase in the hydrolysis time.
The ABTS + free radical scavenging activity of the enzyme hydrolysates of the W. cocos and P. sanguineus increased significantly on increasing the enzymatic hydrolysis time (Figure 8B).
The reducing power of the W. cocos and P. sanguineus increased significantly with an increase in the hydrolysis time ( Figure 8C). The maximum reducing power approached 2.1-fold and 2.9-fold, respectively, after 18 h of hydrolysis.
As shown in Figure 8D, the total phenolic contents of the hydrolysates of the W. cocos and P. sanguineus used herein increased significantly with an increase in the enzymatic hydrolysis time. The P. sanguineus hydrolysate had the higher total phenolic contents after 18 h of hydrolysis.

Evaluation of the Antioxidant Activity of Polyporaceae Hydrolysates
After glucans from the aforementioned the W. cocos and P. sanguineus were hydrolyzed by β-1,3-glucanase for 18 h, the antioxidant activities of enzyme hydrolysates were determined. The free radical scavenging assay revealed a significant increase in the DPPH scavenging potential of the two hydrolysates ( Figure 8A). Low-molecular-weight polysaccharides reportedly display higher antioxidant activity than high-molecular-weight polysaccharides [38][39][40]. Peasura et al. reported that lowmolecular-weight polysaccharides have more reducing ends and can be made to react with free radicals; thus, displaying increased antioxidant activity [41]. The antioxidant ac- Low-molecular-weight polysaccharides reportedly display higher ant ity than high-molecular-weight polysaccharides [38][39][40]. Peasura et al. repo molecular-weight polysaccharides have more reducing ends and can be with free radicals; thus, displaying increased antioxidant activity [41]. The a Low-molecular-weight polysaccharides reportedly display higher anti ity than high-molecular-weight polysaccharides [38][39][40]. Peasura et al. repo molecular-weight polysaccharides have more reducing ends and can be m with free radicals; thus, displaying increased antioxidant activity [41]. The a Wolfiporia cocos. Data: mean ± SE from triplicate experiments (Student's t-test p values: * p < 0.05; ** p < 0.01; *** p < 0.001). The DPPH scavenging potential of P. sanguineus hydrolysates was consistent with an increase in its hydrolysis time. The maximum DPPH scavenging potential approached 2.3-fold after 18 h of hydrolysis. On hydrolyzing W. cocos glucans with β-1,3-glucanase for 1 h, the DPPH scavenging potential peaked and plateaued with an increase in the hydrolysis time.
The ABTS + free radical scavenging activity of the enzyme hydrolysates of the W. cocos and P. sanguineus increased significantly on increasing the enzymatic hydrolysis time ( Figure 8B).
The reducing power of the W. cocos and P. sanguineus increased significantly with an increase in the hydrolysis time ( Figure 8C). The maximum reducing power approached 2.1-fold and 2.9-fold, respectively, after 18 h of hydrolysis.
As shown in Figure 8D, the total phenolic contents of the hydrolysates of the W. cocos and P. sanguineus used herein increased significantly with an increase in the enzymatic hydrolysis time. The P. sanguineus hydrolysate had the higher total phenolic contents after 18 h of hydrolysis.
Low-molecular-weight polysaccharides reportedly display higher antioxidant activity than high-molecular-weight polysaccharides [38][39][40]. Peasura et al. reported that lowmolecular-weight polysaccharides have more reducing ends and can be made to react with free radicals; thus, displaying increased antioxidant activity [41]. The antioxidant activity of fungal polysaccharides is associated with the presence of phenolics [42]. In this study, the total phenolic contents increased as a result of Polyporaceae hydrolysis. The "total phenolic contents" have gained much attention, due to their free radical scavenging abilities and antioxidant activities, which potentially have beneficial implications in human health [43,44]. The "total phenolic contents" is usually considered as one of the important factors for evaluating antioxidant activity. It may suggest the deglycosylation of polyphenols from β-1,3-glucan. This could be confirmed by further experiments. As shown in Figure 7, W. cocos and P. sanguineus had a better hydrolysis rate than L. mylittae and P. umbellatus, with no significant hydrolysis in the latter two Polyporaceae. Although the main polysaccharides in four kinds of Polyporaceae are β-1,3-glucans. However, the content of β-1,3-glucan in various mushrooms is different. The structures of polysaccharides is complex. There are several types of linear or branched glucans in various mushroom species [45]. These differences in content and structure make the degree of hydrolysis different. After 18 h of incubation, enzymatic hydrolysates of the W. cocos and P. sanguineus presented higher total phenolic contents and displayed significantly improved DPPH and ABTS + scavenging activity and reducing power.

Conclusions
Incubation of Y. lipolytica transformant (pYLSC1-13g) with YPD medium for 96 h increased the β-1,3-glucanase activity to 270 U/mL in the culture broth. The transformant β-1,3-glucanase had high thermostability and was tolerant to 10% methanol, ethanol, and DMSO. The transformant β-1,3-glucanase significantly hydrolyzed W. cocos and P. sanguineus. The DPPH and ABTS scavenging abilities, reducing power, and total phenolic contents of the aforementioned two Polyporaceae hydrolysates were significantly increased after 18 h of hydrolysis. This study shows that W. cocos and P. sanguineus glucans hydrolysates produced using the Thermobifida fusca β-1,3-glucanase in a Y. lipolytica transformant (pYLSC1-13g) have high antioxidant activities.