Cloning, Expression, Purification, and Characterization of a Novel β-Galactosidase/α-L-Arabinopyranosidase from Paenibacillus polymyxa KF-1

Glycosidases are essential for the industrial production of functional oligosaccharides and many biotech applications. A novel β-galactosidase/α-L-arabinopyranosidase (PpBGal42A) of the glycoside hydrolase family 42 (GH42) from Paenibacillus polymyxa KF-1 was identified and functionally characterized. Using pNPG as a substrate, the recombinant PpBGal42A (77.16 kD) was shown to have an optimal temperature and pH of 30 °C and 6.0. Using pNPαArap as a substrate, the optimal temperature and pH were 40 °C and 7.0. PpBGal42A has good temperature and pH stability. Furthermore, Na+, K+, Li+, and Ca2+ (5 mmol/L) enhanced the enzymatic activity, whereas Mn2+, Cu2+, Zn2+, and Hg2+ significantly reduced the enzymatic activity. PpBGal42A hydrolyzed pNP-β-D-galactoside and pNP-α-L-arabinopyranoside. PpBGal42A liberated galactose from β-1,3/4/6-galactobiose and galactan. PpBGal42A hydrolyzed arabinopyranose at C20 of ginsenoside Rb2, but could not cleave arabinofuranose at C20 of ginsenoside Rc. Meanwhile, the molecular docking results revealed that PpBGal42A efficiently recognized and catalyzed lactose. PpBGal42A hydrolyzes lactose to galactose and glucose. PpBGal42A exhibits significant degradative activity towards citrus pectin when combined with pectinase. Our findings suggest that PpBGal42A is a novel bifunctional enzyme that is active as a β-galactosidase and α-L-arabinopyranosidase. This study expands on the diversity of bifunctional enzymes and provides a potentially effective tool for the food industry.


Introduction
β-Galactosidases (EC 3.2.1.23),which hydrolyze β-D-galactose residues at the nonreducing end of sugar conjugates, are used in dairy processing [1], oligogalactose synthesis [2], enzyme replacement treatment [3,4], and genetic screening [5].The physical and biological properties of naturally occurring plant cell wall polysaccharides and their corresponding oligosaccharides are of great interest, and many have been used as functional food ingredients [6].β-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose, and also takes part in the transgalactosylation reaction that produces galato-oligosaccharide (GOS) (e.g., Gal (β1→3) Gal (β1→4) Gal (β1→6)) [7,8].Lactose is the most common disaccharide found in mammalian milk.Individuals with lactose intolerance are congenitally unable to decompose lactose and experience various symptoms, such as abdominal pain and diarrhea when consuming dairy products.Approximately 70% of the world population and more than 90% of East Asians suffer from lactose intolerance [9].Therefore, β-galactosidase is widely Molecules 2023, 28, 7464 2 of 15 used to produce low-lactose milk in people with lactose intolerance.β-Galactosidase can also degrade galactan or arabinogalactan to oligosaccharides, which are considered prebiotics [10].α-L-arabinopyranosidases (EC 3.2.1.-)are a class of arabinoglycosidases that break down the non-reducing end of α-L-arabinopyranoside bonds in sugars that contain L-arabinose.α-L-arabinopyranose is a component of many plant polysaccharides and arabinoglycans, and α-L-arabinopyranosidase has potential applications in the biotransformation of these substances [11,12].Ginsenoside Rb2, which is the main components of ginseng (the root of Panax ginseng C.A. Meyer, Araliaceae), is an important medicinal herb in Asia.Ginsenoside Rd has shown an inhibitory effect on carrageenan-induced inflammation, a promotive effect on neural stem cells, and a wound-healing effect [13].Ginsenoside Rd is structurally similar to Rb2, but lacks one outer glycoside moiety at position C20.Therefore, Rb2 can be transformed into Rd by the cleavage of the outer arabinopyranose by α-L-arabinopyranosidase.
Paenibacillus polymyxa is a class of aerobic (or partially anaerobic) bacteria that is widely distributed in nature.This bacterium can grow well in relatively harsh environments with hyperosmolarity, high acidity, high alkalinity, and high or low temperatures [20].Moreover, it produces a variety of extracellular hydrolases in the defense against pathogenic bacteria and is widely used in various industrial applications.The enzymes currently extracted from Paenibacillus polymyxa include the pectinases of the polysaccharide lyase family 9 (PL9) and PL10 families, and the glucanases of the GH5 family [21,22].However, none of the β-galactosidases/α-L-arabinopyranosidases from Paenibacillus polymyxa have been characterized.In the present study, a novel β-galactosidase/α-L-arabinopyranosidase gene (PpBGal42A) of GH42 from Paenibacillus polymyxa KF-1 was cloned and expressed in Escherichia coli, and the recombinant PpBGal42A was purified.The enzymatic properties of PpBGal42A were comparatively characterized in detail to elucidate their feasibilities for application in polysaccharide degradation and in other biotech applications.

Gene Cloning and Analysis of PpBGal42A
The coding cDNA for the gene PpBGal42A from P. polymyxa KF-1 was 2028 bp, as stated in the NCBI (GenBank accession number: WP040101590.1).This cDNA encoded 675 amino acid residues with a pI of 5.23.The theoretical molecular mass of PpBGal42A is 77.16 kDa.We compared PpBGal42A with previously published enzymes (Figure 1) in the GH42 family.PpBGal42A showed the greatest similarity (66% identity) with a β-galactosidase from Bacillus circulans sp.Alkalophilus [23], followed by a 43% similarity to a cold-amplified beta-galactosidase from Rahnella sp.R3 [24], a 38% similarity to Gan42B from Geobacillus stearothermophilus [25], and a 32% similarity to BlGal42A from Bifidobacterium animalis subsp.lactis Bl-04 [26].PpBGal42A has relatively low identity with other-galactosidases. Furthermore, two amino acids, Glu150 and Glu307, were predicted to be the catalytic acid/base and nucleophile, respectively [27].
animalis subsp.lactis Bl-04 [26].PpBGal42A has relatively low identity with other-galactosidases. Furthermore, two amino acids, Glu150 and Glu307, were predicted to be the catalytic acid/base and nucleophile, respectively [27].The 3D-modeled structure of PpBGal42A was constructed using SWISS-MODEL and the structure of the galactosidase from Bacillus circulans sp.Alkalophilus (PDB accession number: 3TTS, identity = 65.82%) as a template [23].The GMQE and QMEAN values for the homology model were 0.91 and 0.89, respectively, indicating a good model quality and high reliability.The structure of the resulting PpBGal42A monomer is shown in Figure 2A, which shows three distinct domains: domain A (residues 1-396), domain B (397-608), and domain C (609-673).The homologous modeling results showed that PpBGal42A preserved the catalytic sites (Asn149, Glu150, Met306, and Glu307).A comparison of PpBGal42A with four other homologous GH42-galactosidase structures reported to date indicated a relatively high structural similarity (Figure 2B).All proteins were composed The 3D-modeled structure of PpBGal42A was constructed using SWISS-MODEL and the structure of the galactosidase from Bacillus circulans sp.Alkalophilus (PDB accession number: 3TTS, identity = 65.82%) as a template [23].The GMQE and QMEAN values for the homology model were 0.91 and 0.89, respectively, indicating a good model quality and high reliability.The structure of the resulting PpBGal42A monomer is shown in Figure 2A, which shows three distinct domains: domain A (residues 1-396), domain B (397-608), and domain C (609-673).The homologous modeling results showed that PpBGal42A preserved the catalytic sites (Asn149, Glu150, Met306, and Glu307).A comparison of PpBGal42A with four other homologous GH42-galactosidase structures reported to date indicated a relatively high structural similarity (Figure 2B).All proteins were composed of three relatively similar domains per monomer; however, although domain A seemed to be very similar between these structures, domains B and C seemed to vary more significantly in their general conformation and in some local loop structures (Figure 2B).
of three relatively similar domains per monomer; however, although domain A seemed to be very similar between these structures, domains B and C seemed to vary more significantly in their general conformation and in some local loop structures (Figure 2B).R-β-Gal (PDB:5E9A, orange) [24], Gan42B (PDB:4OIF, yellow) [25], and BlGal42A (PDB:4UNI, gray) [26].The figure was prepared using Pymol software 2.5.2.

Expression and Purification of Recombinant PpBGal42A
PpBGal42A was cloned into the pET-28a (+) vector and overexpressed in E. coli BL21 (DE3).PpBGal42A was purified using Ni-NTA chromatography and obtained with 30.2 mg from 500 mL of LB medium.The enzyme showed a specific activity of 13.19 U/mg against pNPG.PpBGal42A has a molecular weight of approximately 77 kDa, as assessed by SDS-PAGE (Figure 3).This value is consistent with the predicted molecular weight.
of three relatively similar domains per monomer; however, although domain A seemed to be very similar between these structures, domains B and C seemed to vary more significantly in their general conformation and in some local loop structures (Figure 2B).R-β-Gal (PDB:5E9A, orange) [24], Gan42B (PDB:4OIF, yellow) [25], and BlGal42A (PDB:4UNI, gray) [26].The figure was prepared using Pymol software 2.5.2.

Expression and Purification of Recombinant PpBGal42A
PpBGal42A was cloned into the pET-28a (+) vector and overexpressed in E. coli BL21 (DE3).PpBGal42A was purified using Ni-NTA chromatography and obtained with 30.2 mg from 500 mL of LB medium.The enzyme showed a specific activity of 13.19 U/mg against pNPG.PpBGal42A has a molecular weight of approximately 77 kDa, as assessed by SDS-PAGE (Figure 3).This value is consistent with the predicted molecular weight.

Characterization of Recombinant PpBGal42A
Using pNPG as a substrate, the effect of pH on PpBGal42A activity was investigated in the range pH4.0-11.0.With optimal activity at pH 6.0, the enzyme exhibited good stability at pH 7.0-8.0,and retained >95% activity after 12 h of incubation at 4 • C. The optimal temperature for PpBGal42A was found to be 30 • C, and the PpBGal42A activity decreased sharply when the temperature reached 35 • C. The protein was incubated for 6 h at 20 • C to 40 • C, and maintained >90% activity at temperatures 20-30 • C (Figure 4A).Using pNPαArap as a substrate, the optimal activity was at pH 7.0, and the enzyme exhibited good stability at pH 6.0-9.0 and retained >80% activity.The optimal temperature for PpBGal42A was found to be 40 • C, and maintained >60% activity at temperatures 20-25 • C (Figure 4B).

Characterization of Recombinant PpBGal42A
Using pNPG as a substrate, the effect of pH on PpBGal42A activity was investigated in the range pH4.0-11.0.With optimal activity at pH 6.0, the enzyme exhibited good stability at pH 7.0-8.0,and retained >95% activity after 12 h of incubation at 4 °C.The optimal temperature for PpBGal42A was found to be 30 °C, and the PpBGal42A activity decreased sharply when the temperature reached 35 °C.The protein was incubated for 6 h at 20 °C to 40 °C, and maintained >90% activity at temperatures 20-30 °C (Figure 4A).Using pNPαArap as a substrate, the optimal activity was at pH 7.0, and the enzyme exhibited good stability at pH 6.0-9.0 and retained >80% activity.The optimal temperature for PpBGal42A was found to be 40 °C, and maintained >60% activity at temperatures 20-25 °C (Figure 4B).As shown in Table 1, the effects of metal ions and chemical reagents on the activity of PpBGal42A were investigated.PpBGal42A was weakly activated by Na + , K + , Li + , and Ca 2+ (5 mmol/L).Meanwhile, PpBGal42A was completely inhibited by Mn 2+ , Cu 2+ , Zn 2+ , and Hg 2+ (5 mmol/L), and was significantly inhibited by Fe 2+ and Ni 2+ (5 mmol/L) (Table 1).The Michaelis-Menten parameters for PpBGal42A with pNPG as a substrate were determined as a Km of 1.1 ± 0.2 g/L and Vm of 232.6 ± 9.8 µmol/min/mg.As shown in Table 1, the effects of metal ions and chemical reagents on the activity of PpBGal42A were investigated.PpBGal42A was weakly activated by Na + , K + , Li + , and Ca 2+ (5 mmol/L).Meanwhile, PpBGal42A was completely inhibited by Mn 2+ , Cu 2+ , Zn 2+ , and Hg 2+ (5 mmol/L), and was significantly inhibited by Fe 2+ and Ni 2+ (5 mmol/L) (Table 1).The Michaelis-Menten parameters for PpBGal42A with pNPG as a substrate were determined as a Km of 1.1 ± 0.2 g/L and Vm of 232.6 ± 9.8 µmol/min/mg.

Hydrolysis of Lactose
Lactose was docked into the substrate-binding pocket of PpBGal42A to generate a binding mode.The docking results revealed that lactose bound to the active site pocket of PpBGal42A (Figure 7).Substrate-enzyme interaction analyses were performed to determine the substrate recognition mechanisms.Four AA residues (Pro278, Gln313, Ser311, and Ser320) formed seven hydrogen bonds with lactose.These results show that PpBGal42A has a strong lactose-binding ability, which is beneficial for substrate hydrolysis.The binding energy between PpBGal42A and lactose was −8.2 kcal/mol, which was lower than that of the binding energy (−7.7 kcal/mol) between B. circulans (PDB:3TTS) and lactose.The hydrolysis rate of lactose with PpBGal42A reached about 22.6%, and 82.0% at 4 °C, and 30 °C after 24 h incubation, respectively (Figure 8).

Hydrolysis of Lactose
Lactose was docked into the substrate-binding pocket of PpBGal42A to generate a binding mode.The docking results revealed that lactose bound to the active site pocket of PpBGal42A (Figure 7).Substrate-enzyme interaction analyses were performed to determine the substrate recognition mechanisms.Four AA residues (Pro278, Gln313, Ser311, and Ser320) formed seven hydrogen bonds with lactose.These results show that PpBGal42A has a strong lactose-binding ability, which is beneficial for substrate hydrolysis.The binding energy between PpBGal42A and lactose was −8.2 kcal/mol, which was lower than that of the binding energy (−7.7 kcal/mol) between B. circulans (PDB:3TTS) and lactose.The hydrolysis rate of lactose with PpBGal42A reached about 22.6%, and 82.0% at 4 • C, and 30 • C after 24 h incubation, respectively (Figure 8).

PpBGal42A and Pectinase Display Synergistic Activity in Degrading Citrus Pectin
When citrus pectin was incubated with pectinase for 24 h, we observed that ~579 µg of reducing sugar was released.Meanwhile, the incubation of citrus pectin with PpBGal42A for 24 h released ~3.5 µg of reducing sugar.However, when both enzymes were combined, 642 µg of reducing sugar was released from citrus pectin, which is a 1.1fold increase compared to their individual use.This suggested a relatively modest synergistic effect (Table 3).

PpBGal42A and Pectinase Display Synergistic Activity in Degrading Citrus Pectin
When citrus pectin was incubated with pectinase for 24 h, we observed that ~579 µg of reducing sugar was released.Meanwhile, the incubation of citrus pectin with PpB-Gal42A for 24 h released ~3.5 µg of reducing sugar.However, when both enzymes were combined, 642 µg of reducing sugar was released from citrus pectin, which is a 1.1-fold increase compared to their individual use.This suggested a relatively modest synergistic effect (Table 3).

Discussion
Paenibacillus polymyxa is a potentially important biotechnological agent [20], because it efficiently produces an array of compounds that are useful in industrial processes.In this study, we cloned and expressed the β-galactosidase/α-L-arabino-pyranosidase gene PpB-Gal42A from Paenibacillus polymyxa KF-1 with an expression level of up to 60.4 mg/L and specific activity towards pNPG (13.19 U/mg).SDS-PAGE showed that purified PpBGal42A appeared as a single band with a relative molecular mass of ~77 kDa.
Using pNPG as substrate, the optimal pH for PpBGal42A activity was pH 6.0, and ~95% activity was maintained in the pH range of 7-8.β-galactosidase.A pH between 6-7.5 is suitable for hydrolyzing the lactose present in milk and sweet whey [4].The optimal temperature for PpBGal42A was observed to be 30 • C, with ~90% activity being maintained at temperatures ranging from 20 • C to 30 • C. Using pNPαArap as substrate, the optimal temperature was 40 • C, the optimal activity was at pH 7.0, and the enzyme exhibited good stability at pH 6.0-9.0, and retained >80% activity.PpBGal42A exhibited a relatively higher temperature and pH stability.The pH and temperature ranges support the idea that PpBGal42A is a potentially useful bioindustrial tool.Enzymes generally face unfavorable reaction conditions when used in industrial bioprocesses, and thus must have the ability to tolerate various reaction conditions.The stability of β-galactosidase was investigated in the presence of various metal ions and surfactants.Mono-and divalent cations affect β-galactosidase activity [28].K + and Na + enhance the activity of Gal3149 from Bacillus velezensis SW5, while Zn 2+ and Cu 2+ strongly or completely inhibit its activity [29].The divalent cations Mg 2+ , Ca 2+ , and Zn 2+ were found to enhance the catalytic activity of BgaC, whereas Cu 2+ and Mn 2+ were inhibitory [30].In this study, PpBGal42A was slightly activated by Na + , K + , Li + , and Ca 2+ (5 mmol/L).PpBGal42A was significantly or completely inhibited by 5 mM Mn 2+ , Cu 2+ , Zn 2+ , Hg 2+ Fe 2+ , or Ni 2+ .
We also studied the ability of PpBGal42A to degrade lactose.Lactose is partially degraded to galactose and glucose at 30 • C or 4 • C and may be completely degraded when the amount of PpBGal42A is increased or when the reaction time is prolonged.This feature enables the use of this enzyme for the removal of lactose from dairy products.The hydrolysis of lactose in milk can be performed chemically or enzymatically.β-Galactosidase is widely used in the production of lactose-free dairy products because it avoids the production of byproducts and does not alter the physicochemical properties of milk [16,36].These products are intended for consumption by lactose-intolerant patients whose digestive systems are deficient in β-galactosidase [37].In addition, β-galactosidase is used to prepare ice cream and condensed milk to avoid lactose crystallization and enhance the sweetness and creaminess of these products [38].
Our in-depth analysis of the hydrolytic characteristics of PpBGal42A supports its use in specific instances.For example, PpBGal42A exhibits significant degradative activity towards citrus pectin when combined with pectinase.Our findings underscore the significant benefits of this pair of enzymes when delineating structure-function relationships in polysaccharides and their biological functions.We expect that PpBGal42A will be an excellent candidate for the identification of fruit juice pectin.

Construction of Plasmids and Strains
Total DNA was extracted from P. polymyxa KF-1 using a DNA Extraction Kit.Two primers, forward (5 -GACTGGTGGACAGCAAATGGGTCGCGGATCCA-TGATAAGCAGCAAACT TCC-3 ) and reverse (5 -GATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTAGGATAGCTC CAGCACTT-3 ) that contain restriction sites for BamHI and XhoI (restriction sites underlined), respectively, were designed based on the gene.PCR was performed using 2 × Taq PCR Green Mix with the following protocol: 94 • C for 3 min, 30 cycles at 94 • C for 30 s, 56 • C for 30 s, 72 • C for 2 min, and finally at 72 • C for 5 min.The PCR product and pET-28a (+) were digested with BamHI and XhoI and ligated with pET-28a (+) to generate the recombinant plasmid pET-28a-PpBGal42A.All enzymes were obtained from New England Biolabs (Beverly, MA, USA).Restriction enzyme digestion, ligations, and transformations were performed according to the supplier's recommendations.

Expression and Purification of Recombinant PpBGal42A
By heat shock, pET-28a-PpBGal42A was transformed into E. coli BL21 (DE3).The positive transformants were verified by DNA sequencing (Sangon Biotech, Shanghai, China).The E. coli transformants were grown in LB medium supplemented with 50 µg/mL kanamycin.When the optical density at 600 nm reached 0.6-0.8,IPTG (final concentration of 0.5 mM) was added, and the incubation was continued at 16 • C for 20 h, followed by centrifugation at 8000 rpm for 10 min to collect cells and ultrasonically disrupt them.linear arabinosaccharides (LSA)) were used to evaluate the hydrolytic activities of PpB-Gal42A.After 24 h of reaction at 30 • C, samples were tested for molecular weight changes using TSK-G3000.The released products were detected by HPAEC using a CarboPac PA-200 column (3 × 250 mm) attached to a Dionex ICS-5000 Plus ion chromatographic system, using the protocol: 0-10 min, 50 mM NaOH; 10-30 min linear gradient of 0-100 mM NaAc in 5 mM NaOH; 30-40 min, 500 mM NaAc in 200 mM NaOH [44].

Synergistic Action of PpBGal42A with Pectinase with Citrus Pectin
Degradation of citrus pectin was performed by incubating 1 mg/mL citrus pectin with PpBGal42A or pectinase (or both) in 20 mM NaAc-HAc buffer (pH 6.0) at 30 • C for 24 h.The amount of reducing sugar released was measured using the DNS method [45], and comparing it to a standard curve generated using galactose.

Figure 4 .
Figure 4. Effect of pH and temperature on activity and stability of PpBGal42A.(A) Using pNPG as substrate.(A-a) Optimal pH.(A-b) pH stability.(A-c) Optimal temperature.(A-d) Temperature stability.(B) Using pNPαArap as substrate.(B-a) Optimal pH.(B-b) pH stability.(B-c) Optimal temperature.(B-d) Temperature stability.Relative activity was calculated using the maximum activity as 100%.Results are presented as the mean ± standard deviation (n = 3).

Figure 4 .
Figure 4. Effect of pH and temperature on activity and stability of PpBGal42A.(A) Using pNPG as substrate.(A-a) Optimal pH.(A-b) pH stability.(A-c) Optimal temperature.(A-d) Temperature stability.(B) Using pNPαArap as substrate.(B-a) Optimal pH.(B-b) pH stability.(B-c) Optimal temperature.(B-d) Temperature stability.Relative activity was calculated using the maximum activity as 100%.Results are presented as the mean ± standard deviation (n = 3).

Figure S2 :
Mw of PpBGal42A determined by SDS-PAGE; Table S1: Summary of expression and purification of recombinant PpBGal42A.Author Contributions: J.C.: conceptualization, investigation, writing-original draft.Y.W., A.Z. and S.H.: methodology, project administration.Z.M.: supervision.T.C.: visualization.N.W.: software.Y.Y.: writing-review and resources.All authors have read and agreed to the published version of the manuscript.Funding: This work was funded by the Scientific and Technologic Foundation of Jilin Province (No. 20200201190JC), the Jilin Province Development and Reform Commission (No. 2022C041-1), and the National Natural Science Foundation of China (No. 32000907).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Influence of different chemicals on the β-galactosidase activity of PpBGal42A.

Table 1 .
Influence of different chemicals on the β-galactosidase activity of PpBGal42A.
Results are presented as the mean ± standard deviation (n = 3).

Table 2 .
Determination of specific activities for recombinant PpBGal42A with nitrophenyl-linked substrates.

Table 3 .
Degradation of pectin in combination with PpBGal42A and pectinase.

Table 3 .
Degradation of pectin in combination with PpBGal42A and pectinase.