Site-directed mutation of β-galactosidase from Streptococcus thermophilus for galactooligosaccharide-enriched yogurt making

β-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the β-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify β-galactosidase from Streptococcus thermophilus . Out of 28 Strep. thermophilus strains, a β-galactosidase gene named bgaQ , encoded for high β-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr 801 and Pro 802 around the active sites of BgaQ with His 801 and Gly 802 , the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bul-garicus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of β-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.


INTRODUCTION
Yogurt, one of the most popular fermented dairy products, is typically fermented using 2 starter cultures, from Streptococcus thermophilus and Lactobacillus delbrueckii ssp.bulgaricus (Horiuchi et al., 2009).However, most yogurt starter cultures only metabolize the glucose moiety of lactose and release the galactose in the extracellular medium, resulting in significant amounts of galactose accumulation (Hols et al., 2005;Zhang et al., 2020b).Lactose is not recommended for individuals with symptom of lactose intolerance, which is a common disease in more than half of the world's population (Husain, 2010).In addition, amounts of galactose also reduce the quality of fermented dairy products.For low-sugar yogurt making, several attempts have been made to isolate galactose-positive wild-type Strep.thermophilus strains or to co-culture starter cultures with other Lactobacillus species (Robitaille et al., 2007;Anbukkarasi et al., 2014;Sørensen et al., 2016;Zhang et al., 2020b).Also, arabinose isomerase has been used during milk fermentation to convert galactose into the low-calorie sweetener tagatose (Rhimi et al., 2011).
Streptococcus thermophilus is one of the most important starters for fermented dairy products with high β-galactosidase hydrolysis activity (Anbukkarasi et al., 2014;Zhang et al., 2020a).However, the transgalactosylation activity of the enzyme in Strep.thermophilus is normally low, resulting in galactose accumulation in yogurt.In this study, a β-galactosidase BgaQ was isolated from Strep.thermophilus SDMCC050237.After the prediction of structural and active sites via bioinformatics analysis, genetic modification of BgaQ through a site-directed mutagenesis was performed, and the GOS biosynthesis of the generated mutated enzyme in 5% lactose and reconstituted skim milk (RSM) medium were determined.The aim of this study was to improve the transgalactosylation activity of β-galactosidase from Strep.thermophilus, to catalyze galactose into GOS for manufacture of GOS-enriched yogurt.

Bacterial Strains, Plasmids, and Cultivation Conditions
The strains and plasmids used in this study are listed in Supplemental Table S1 (https: / / zenodo .org/record/ 5720718 # .YZzi9sVBw2w).Streptococcus thermophilus strains were cultured in M17 broth (Oxoid) supplemented with 1% lactose at 42°C.The Lb. bulgaricus strain ATCC11842 was grown statically in de Man, Rogosa, and Sharpe medium at 37°C.Escherichia coli strains were grown aerobically in Luria-Bertani medium at 37°C.If necessary, kanamycin was supplemented to a final concentration of 30 μg/mL.

Determination of Hydrolysis and Transgalactosylation Activities of β-Galactosidases from Strep. thermophilus
Cells of Strep.thermophilus strains cultured in lactose-supplemented M17 broth at 42°C for 12 h were harvested by centrifugation at 6,000 × g for 5 min at 4°C, washed twice with PBS (pH 7.4), and suspended in PBS.The cells were disrupted by a cell homogenizer and centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was used to assay the hydrolysis and transgalactosylation activities of the β-galactosidases as described previously (Lu et al., 2015;Xin et al., 2019).Briefly, the hydrolysis activity of the β-galactosidase was performed in 200-μL mixtures containing 50 μL of the cell supernatant and 150 μL of lactose solution (200 g/L) dissolved in PBS (pH 7.4), and incubated at 50°C for 10 min.Reactions were stopped by heating at 100°C for 5 min.The amount of glucose released in the reaction mixture was detected using a glucose assay kit (Robio).One unit of β-galactosidase activity was defined as the amount of enzyme releasing 1 mmol of d-glucose per minute.
The transgalactosylation reaction was performed in 100-μL mixtures containing 50 μL of the cell supernatant and 50 μL of lactose solution (200 g/L) dissolved in PBS (pH 7.4) at 42°C for 4 h, and then stopped by heating at 100°C for 5 min.Five microliters of the reaction solution was spotted onto thin-layer chromatography plates (Merck).The sugars were separated by a developing solvent consisting of n-butanol, isopropyl alcohol, acetic acid, and water (38.9%:27.8%:11.1%:22.2%).Subsequently, the plates were air-dried and sprayed with a chromogenic agent consisting of acetone-anilinephosphoric acid (50:1:5 by volume) containing 0.018% (wt/vol) diphenylamine.After incubation at 105°C for 5 min, the bands were visualized and then photographed (Lu et al., 2015).

Molecular Manipulation
All molecular manipulations in this study were performed as described previously (Xin et al., 2019).Plasmids from E. coli were extracted using a Plasmid Mini Kit (Omega).Taq polymerase, restriction enzymes, and T4 DNA ligase were purchased from TaKaRa and used according to the standard procedures.
For the biochemical property analysis of β-galactosidase, the gene bgaQ was PCR amplified from the genomic DNA of Strep.thermophilus SD-MCC050237 with primers bgaQ F (5′-CATGCCATG-GACATGACTGAAAAAATTCAAA-3′; underlining indicates the Nco I site) and bgaQ R (5′-CCGCTC-GAGATTTAGTGGTTCAATCATGAAGCTT-3′; underlining indicates the Xho I site).The PCR product was digested with the restriction enzymes Nco I and Xho I and then ligated into the corresponding sites of plasmid pET28a, generating the recombinant plasmid pET28a-bgaQ.After transformation into E. coli BL21 (DE3), the resulting recombinant E. coli BL21 carrying BgaQ was cultured at 37°C until cultures' optical density at 600 nm reached 0.6; then 0.1 mM isopropyl-β-dthiogalactoside was added to the medium, and culturing continued for 20 h at 16°C for protein expression.Then the cells of E. coli BL21 carrying BgaQ were harvested by centrifugation at 6,000 × g for 5 min at 4°C and washed twice with PBS.The cells were disrupted by ultrasonic treatment and centrifuged at 10,000 × g for 20 min at 4°C to remove cell debris.The expression of the protein BgaQ was determined on SDS-PAGE gel after purification by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, according to standard procedures.

Sequence Analysis and Protein Modeling of the β-Galactosidase BgaQ
Sequence identity and similarity searches for the gene bgaQ were performed using Blast at NCBI (http: / / www .blast.ncbi.nlm.nih.gov).Multiple-sequence alignment of BgaQ with other related β-glucosidase sequences was performed using ClustalX 2.0 (Larkin et al., 2007).The 3-dimensional structure of BgaQ was generated using the Swiss-Model protein modeling server (http: / / swissmodel .expasy.org).The topology structure of β-galactosidase from E. coli (Protein Data Bank identification number 1PX3) was obtained from the RCSB Protein Data Bank (http: / / www .rcsb.org).The modeled structures were analyzed for the active sites of BgaQ by the Pymol molecular graphics system (http: / / www .pymol.org).The amino acid sites associated with transgalactosylation activity were found by autodocking using β-3′-galactosyl-lactose as substrate.

Site-Directed Mutagenesis of the β-Glucosidase BgaQ
To genetic modify the transgalactosylation activity, the Tyr 801 and Pro 802 around the activity sites in BgaQ sequence were predicted and replaced by His 801 and Gly 802 using overlap PCR with the mutagenic primer pairs 8012-forward 5′-ACAACGATCGTGGTGCTG-GACATGGATTCGAAATG-3′ and 8012-reverse 5′-TCCAGCCTGCCATTTCGAATCCATGTCCAG-CACCA-3′, with the underlined nucleotides coding the amino acids His and Gly at positions 801 and 802.After digestion with NcoI and XhoI, the PCR products were ligated into the corresponding sites of the plasmid pET28a, generating the recombinant plasmid pET28-bgaQ-8012, the mutant protein BgaQ-8012 was expressed in E. coli BL21 (DE3) and purified as previously described for BgaQ.

Biochemical Properties of BgaQ and Mutant BgaQ-8012
The optimal temperature for hydrolysis activities of the purified β-galactosidase BgaQ and the mutant BgaQ-8012 were determined over the range of 25 to 65°C.For the thermostability assay, the residual activities of the purified enzymes were measured after incubation from 37 to 60°C for 100 min.Three buffer systems, citrate buffer (50 mM, pH 5.5), sodium phosphate buffer (50 mM, pH 6.0-8.0), and Tris-HCl buffer (50 mM, pH 8.5-10.0),were used to measure the optimal pH for hydrolysis activity of the β-galactosidase BgaQ and the mutant BgaQ-8012 at 42°C.The residual activities were measured for pH stability in various buffers from pH 6.0 to 10.0.For optimization of GOS synthesis conditions, pH from 6.0 to 8.0 and reaction temperatures from 37 to 60°C were adopted.

GOS Biosynthesis with BgaQ and BgaQ-8012
To simulate the fermentation conditions of yogurt processing, GOS production was measured at 5% lactose dissolved in PBS (pH 6.5) at 42°C, containing 1 U of purified BgaQ or BgaQ-8012, for 240 min.Samples were collected at intervals of 40 min.The GOS in the samples were analyzed by HPLC using an Aminex HPX-87H Carbohydrate Column (300 mm × 7.8 mm; Bio-Rad) at 65°C, with a refractive index detector.The mobile phase was deionized water at a flow rate of 0.5 mL/min.The GOS yield was calculated from the total amount of saccharides eluted at the retention times.

Fermented Milk
BgaQ and BgaQ-8012 were applied to catalyze GOS synthesis in milk.An RSM medium was prepared by reconstituting skim milk powder (10% wt/vol) with distilled water and sterilized at 115°C for 10 min.The 2 starters Strep.thermophilus SDMCC050237 and Lb.bulgaricus ATCC11842 were cocultured at a ratio of 4:1 in 100 mL of RSM medium to which 1 U of the purified BgaQ or BgaQ-8012 was added; fermentation without purified β-galactosidase was used as control.After incubation at 42°C for 12 h, the curd time was observed visually, indicating the time at which milk changed from a flowing liquid to an immobile semisolid state.The concentrations of lactose, galactose, and GOS in yogurt were determined via HPLC.The pH, titratable acidity, syneresis, and viable cell counts were detected at the end of fermentation, as described by da Costa et al. ( 2016) and Li et al. (2017).

Statistical Analysis
Each experiment was carried out in triplicate, and data are presented as mean ± standard deviation.Statistical significance between yogurts fermented by traditional starters with or without β-galactosidase was determined using SPSS version 19.0 (IBM Corp.), with P < 0.05 considered as statistically significant.

Activities of β-Galactosidases from Strep. thermophilus
The hydrolysis and transgalactosylation activities of the β-galactosidases from 28 Strep.thermophilus strains were determined.Results presented in Figure 1 showed that the hydrolysis activities of β-galactosidases occurred in a strain-specific manner among the Strep.thermophilus strains tested, and the strain Strep.thermophilus SDMCC050237 exhibited the highest hydrolysis activity, reaching 1.33 U/mL.However, the transgalactosylation activities of the β-galactosidases from the tested Strep.thermophilus strains appeared relatively low, as observed by small amounts of GOS production (Figure 2).Therefore, the β-galactosidase (BgaQ) from Strep.thermophilus SDMCC050237 was selected for the following analysis.
To improve the transgalactosylation activity of the BgaQ, the catalyzing domain of this enzyme was predicted by comparison with that of the known β-galactosidase 1PX3 from E. coli.As shown in Figure 3a, the 2 active sites of glutamate, Glu 458 and Glu 546 , were identified in BgaQ, and 10 potential transgalactosylation-related sites around them were provided by autodocking using β-3′-galactosyl-lactose as substrate (Figure 3a).Sequence alignment of the BgaQ with the high transgalactosylation activities of the β-galactosidases from other bacteria showed that the amino acid residues Tyr 801 and Pro 802 around the active sites Glu 458 and Glu 546 varied significantly in specific strains (Figure 3b), leading us to speculate that the sites Tyr 801 and Pro 802 were the key sites affecting the transglycosylation activity of the enzyme BgaQ.Thus, the amino acid residues Tyr 801 and Pro 802 were selected as targets for the site-directed mutagenesis.After replacing the Tyr 801 and Pro 802 with His 801 and Gly 802 by overlap PCR, and detecting the transgalactosylation activity, the mutant protein BgaQ-8012 was obtained.

Biochemical Characterization of BgaQ and BgaQ-8012
For biochemical analysis, BgaQ and BgaQ-8012 were expressed in E. coli DE3 and purified by Ni-NTA Column (Qiagen), respectively.The 2 purified protein bands appeared the same size, approximately 113 KD, on SDS-PAGE gel, which was in accordance with their theoretical molecular mass (Figure 4).Meanwhile, no statistical difference was detected between the hydrolytic activities of these 2 purified β-galactosidases.The optimum temperature and pH of the purified BgaQ-8012 were also the same as those of the wild-type en-zyme BgaQ at 50°C and 8.5, respectively (Figure 5).In addition, BgaQ-8012 was shown to be more sensitive to temperature, with only 19% hydrolytic activity at 50°C for 40-min incubation, whereas BgaQ retained 57% activity under the same conditions (Figure 6).For pH stability, both BgaQ and BgaQ-8012 were more stable in acidic pH, showing maximal stability at pH 6.5.
The transgalactosylation conditions of these 2 purified proteins were detected.As shown in Figure 7a, the optimal pH for both BgaQ and BgaQ-8012 was 6.5.However, the optimal temperature showed obvious difference, with the optimal temperature of BgaQ being 50°C and 42°C for BgaQ-8012 (Figure 7b).

GOS Yields at 5% Lactose
To simulate the lactose content of yogurt processing, GOS production was performed at 5% lactose dissolved in PBS (pH 6.5) at 42°C, containing purified BgaQ or BgaQ-8012, for 240 min.Results showed that the GOS yields in these 2 reaction mixtures increased with prolonged time.The GOS yield of BgaQ-8012 was obviously higher than that of BgaQ during the reaction processes: the maximal GOS yield of BgaQ was about 20.5% at 120 min, compared with 26.7% for BgaQ-8012  at 160 min, indicating that the transgalactosylation activity of the BgaQ increased with the site-directed mutagenesis (Figure 8).

Properties of Yogurt Fermented with BgaQ and BgaQ-8012
Purified BgaQ or BgaQ-8012 were added to the RSM medium in which 2 traditional starters, Strep.thermophilus SDMCC050237 and Lb.bulgaricus ATCC11842, were inoculated.After incubation at 42°C for 12 h, the contents of lactose and total sugar were significantly reduced by 38.6% and 16.5% (incubated with 2 traditional starters), 49.3% and 26.5% (incubated with 2 traditional starters+ BgaQ), and 54.4% and 40.1% (incubated with 2 traditional starters + BgaQ-8012), respectively (Table 1).Meanwhile, the GOS yields in yogurt fermented with 2 traditional starters and with added BgaQ and BgaQ-8012 were 3.7 g/L, 5.8 g/L, and 8.3 g/L (Table 1).The GOS yields of the mutant enzyme BgaQ-8012 increased compared with the wild enzyme BgaQ (Table 1).However, the galactose contents in all samples increased, particularly in the yogurts with the added β-galactosidase, BgaQ and BgaQ-8012, suggesting that the low pH in yogurt processing or low lactose content limited the GOS synthesis of the β-galactosidases.
Fermented milk performance analysis showed that the addition of the β-galactosidase BgaQ or BgaQ-8012 shortened the curd time significantly compared with the traditional yogurt starters, and there was no significant difference in pH, titratable acidity, and water-holding capacity of yogurt between them (Table 2).In addition, the viable cell counts of Strep.thermophilus SD-MCC050237 and Lb.bulgaricus ATCC 11842 in all the groups reached 8 log cfu/mL (Supplemental Table S2, https: / / zenodo .org/record/ 5720726 # .YZzk9sVBw2w).

DISCUSSION
β-Galactosidase plays a main role in the conversion of lactose into glucose and galactose in dairy products (Sangwan et al., 2015;Lu et al., 2020), as well as in the production of GOS based on its transgalactosylation property (Liu et al., 2017;Rico-Díaz et al., 2017).Most studies have focused on GOS biosynthesis from high concentrations of lactose (Song et al., 2013;Yu and O'Sullivan, 2014;Sangwan et al., 2015;Cao et al., 2019), while little attention has been paid to GOS production in fermented milk, where the low lactose   mutagenesis from the β-galactosidase BgaQ in Strep.thermophilus SDMCC050237, showed high GOS synthesis ability using 5% lactose as substrate, as well as in yogurt processing.Therefore, this study provides an efficient strategy to improve the transgalactosylation activity of β-galactosidase and could be used for GOSenriched yogurt making.
Site-directed mutagenesis is an invaluable technique to modify genes for the analysis of the structural and functional properties of proteins.Several enzyme variants have been successfully obtained using this strategy, which display enhanced transgalactosylation activity of β-galactosidase or thermal stability compared with the corresponding wild-type enzymes (Yang et al., 2015;Rico-Díaz et al., 2017).But the key of this technique is to identify the targeting mutated sites.Bioinformation software and internet sites provide the possibility of prediction and analysis of the active sites of enzymes.Using these tools, the potential transgalactosylation-related sites of Tyr 801 and Pro 802 around the active sites of Glu 458 and Glu 546 in BgaQ were identified (Figure 3a).On the basis of results reported previously, indicating that aromatic amino acids around the active sites can reduce the efficiency of enzyme binding to the receptor substrate (Bultema et al., 2014), the aromatic amino acid Tyr 801 , which binds to the acceptor substrate, was replaced by His 801 .In consideration of the doubledisplacement reaction of transgalactosylation, including the formation of a glycosylated enzyme reaction intermediate and its deglycosylation either by water molecule (hydrolysis) or by other acceptor (transgalactosylation), it was logical that a reduction in hydrolysis would be beneficial for the transgalactosylation reaction (Qin et al., 2019).Therefore, the amino acid Pro 802 was substituted by Gly 802 , which could be achieved by reducing the water activity around the active sites.As a result, no obvious difference in hydrolytic activities appeared between BgaQ and the mutant BgaQ-8012.As expected, the transgalactosylation activity of the mutant BgaQ-8012 was enhanced, with GOS yield from 20.5% to 26.7% in 5% lactose as substrate (Figure 8), which was in agreement with previous reports (Bultema et al., 2014;Qin et al., 2019).Meanwhile, the optimal temperature for GOS production decreased from 50°C to 42°C, a promising temperature for yogurt fermentation.These results further demonstrate that site-directed mutagenesis is an effective tool for improvement of the transgalactosylation activity of β-galactosidase in Strep.thermophilus.
To investigate the GOS synthesis of the mutant enzyme BgaQ-8012 in yogurt processing, the purified enzymes were added to RSM medium in combination with the 2 traditional starters Strep.thermophilus SD-MCC050237 and Lb.bulgaricus ATCC 11842.As anticipated, the addition of BgaQ or BgaQ-8012 improved the GOS yield, suggesting that the β-galactosidases  from Strep.thermophilus have abilities to synthesize GOS at low levels of lactose content.In addition, the GOS yield of the mutant enzyme BgaQ-8012 increased from 5.8 g/L to 8.3 g/L compared with the wild-type enzyme BgaQ (Table 1), which indicated that the mutant enzyme BgaQ-8012 was more suitable for GOSenriched yogurt making.Compared with the addition of artificial GOS to yogurt directly, the addition of BgaQ or BgaQ-8012 also significantly reduced the contents of lactose and total sugar, as has been previously reported (Ruiz-Matute et al., 2012;Rodriguez-Colinas et al., 2014).Acidification and curding in a short incubation time are crucial for yogurt making.The fermented milk performance analysis showed that the addition of the BgaQ and BgaQ-8012 shortened the curd time in yogurt processing, due to the β-galactosidases BgaQ and BgaQ-8012 added exogenously to accelerate the hydrolysis of lactose into galactose and glucose, which rapidly promoted the growth of the 2 traditional starters Strep.thermophilus SDMCC050237 and Lb.bulgaricus ATCC11842 at the beginning of fermentation processing, produced more lactic acid to lower the pH, and subsequently facilitated the acidification processes.It has been reported that cocultures of Lb. bulgaricus 2038 and Strep.thermophilus 1131 in lactosehydrolyzed milk resulted in increased cell numbers of Lb. bulgaricus 2038 (Yamamoto et al., 2021), but no obvious differences of the cell counts in yogurts with or without addition of the purified enzymes were observed in our study (Supplemental Table S2, https: / / zenodo .org/record/ 5720726 # .YZzk9sVBw2w).Along with the growth of the 2 starters Strep.thermophilus SDMCC050237 and Lb.bulgaricus ATCC11842, the pH dropped quickly, and the lactose content in milk was reduced, which might inhibit the enzyme activities of the β-galactosylases BgaQ and BgaQ-8012, in turn, with more galactose accumulation in the medium.However, the lactose and total sugar contents were decreased obviously; this phenomenon was also as described previously (Oliveira at al., 2011;Ruiz-Matute et al., 2012;Rodriguez-Colinas et al., 2014).Therefore, exogenous addition of the β-galactosidases from Strep.thermophilus, with high transgalactosylation activity, contributed to GOS-enriched yogurt making.Different superscripts indicate significant differences (P < 0.05) between yogurt fermented by the 2 traditional starters with BgaQ and BgaQ-8012.
Figure 4. SDS-PAGE analysis of the purified BgaQ (the β-galactosidase from Streptococcus thermophilus SDMCC050237) and Bga-8012 (the mutant enzyme of BgaQ).M = protein marker; T = total proteins in the lysate.