Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene ( lacLM ) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60℃ and 7.0, and 50℃ and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit. β-D-Galactosidases (β-D-galactoside galactohydrolase or lactase, E.C. 3.2.1.23) catalyze the hydrolysis of lactose to glucose and galactose [24] . After weaning, most people lose 75-90% of their birth lactase level. Clinically, if lactose maldigestion causes physiological symptoms, the person is said to have lactose intolerance [5] . Lactases have been successfully used for the hydrolysis of lactose in dairy products, thus increasing the usability of these foodstuffs, especially for those suffering from lactose intolerance [14 , 23] . Furthermore, β-D-galactosidases can also transfer galactose residues to form galacto-oligosaccharides (GOS) or alkyl glycosides [7 , 25] . This property has given the enzyme widespread use in food industries for the production of GOS as probiotic food stuffs [8 , 22] . The β-galactosidases are classified into four glycoside hydrolase (GH) families: GH1, GH2, GH35, and GH42 (carbohydrate-active enzymes database; http://www.cazy.org ) [21] . Galactosidases in the GH1, GH2, and GH42 families have been found predominantly in bacteria [2 , 6 , 11] , whereas the enzymes in GH35 have been found not only in bacteria but in fungi, animals, and plants as well [9] . Among those galactosidases, one type of β-galactosidase that belongs to the GH2 family has shown an excellent ability to hydrolyze lactose and synthesize galacto-oligosaccharide [18] . Interestingly, this type of β-galactosidase is heterodimeric, consisting of a large subunit (encoded by the lacL gene) and a small subunit (encoded by the lacM gene). Nguyen et al. [17] purified the large and small subunits of β-galactosidase from L. reuteri L103 and found that the large subunit exhibits hydrolytic activity on 4-methylumbelliferyl β-D-galactoside, but the small subunit does not have any activity. Unfortunately, when the lacL gene alone was cloned into pET21d and heterologously expressed in E. coli , β-galactosidase activity failed to be detected. It was supposed that the small subunit plays an important role in the activity of the large subunit. However, the evidence is absent. Although many β-galactosidase genes have been isolated from Lactobacillus thus far, such as L. fermentum [12] , L. sakei [20] , L. coryniformis [3] , and L. acidophilus [16] , these studies focused on the application of β-galactosidase [13 , 17 , 19] . Few studies have reported the relationship between the two subunits of β-galactosidase. Kefir grain collected from Tibet, China, is used locally to make traditional yogurt, which is beneficial to human health [26] . In our laboratory, Lactobacillus kefiranofaciens ZW3, a novel strain, was isolated and identified from kefir grain. Considering its outstanding characteristics, the genomic DNA of L. kefiranofaciens ZW3 was sequenced, the GenBank accession number of which is CP002764.1. Among its characteristics, the strain displays a strong ability to hydrolyze lactose. Therefore, in the present study, we cloned a heterodimeric LacLM-type β-galactosidase gene and its two subunit genes, lacL and lacM , from L. kefiranofaciens ZW3. Furthermore, the three genes were heterologously expressed in Escherichia coli BL21(DE3) and then these recombinant proteins were purified. The character of the LacLM-type β-galactosidase from L. kefiranofaciens ZW3 and the relationship between the large and small subunits were investigated. All of the restriction enzymes, Pfu Taq DNA polymerase, agarose gels, DNA purification kit, and T4 DNA ligase were purchased from Takara Biotechnology Co., Ltd (Dalian, China). A Bacterial Gen DNA kit, and a nickel-nitrilotriacetic acid (Ni-NTA) His-Bind Purification kit were purchased from Cowin Biotech Co., Ltd. (Beijing, China). Isopropyl-β-D-thiogalactopyranoside (IPTG) and o -nitrophenyl-β-galactopyranoside (ONPG) were purchased from Sigma Chemical Company (St. Louis, MO, USA). L. kefiranofaciens ZW3 was obtained from kefir grains and identified by the China General Microbiological Culture Collection (CGMCC, Beijing, China). E. coli DH5α strain was used for DNA manipulations and amplification. E. coli BL21(DE3) was used as the host for the recombinant plasmid harboring pET-30a (+) (Novagen, USA) and for protein expression. Recombinant DNA techniques, including plasmid extraction, restriction endonuclease digestion, and DNA ligation, were performed using standard methods. The L. kefiranofaciens ZW3 strain was grown at 37℃ for 3 days in MRS broth containing peptone 10 g/l, dipotassium hydrogen phosphate 2 g/l, meat extract 8 g/l, diammonium hydrogen citrate 2 g/l, yeast extract 4 g/l, sodium acetate 5 g/l, magnesium sulfate 0.2 g/l, Tween-80 1 g/l, and manganese sulfate 0.04 g/l. Then, the genomic DNA was extracted from L. kefiranofaciens ZW3 using a Bacterial Gen DNA kit. Based on the sequence of lacLM in GenBank (GI: 333957179, 333957180), specific primers were designed and synthesized ( Table 1 ). These genes were amplified by polymerase chain reaction (PCR) from the genomic DNA. The PCR conditions were as follows: a hot start at 94℃ for 5 min, 35 repeated cycles of 94℃ for 30 sec, 62℃ for 30 sec, and 72℃ for 2-3 min, followed by one cycle of 72℃ for 10 min. The PCR products were purified from agarose gels. The purified DNA fragments were ligated to pEASY-Blunt (Transgen Biotech. Co., China) and the plasmids were transformed into E. coli DH5α cells. The resulting recombinant plasmids were isolated from a positive clone and sequenced. The genes were excised from the pEASY-Blunt recombinant plasmids using one pair of restriction enzymes, BamHI and XhoI, and ligated with the pET-30a(+) vector, which was digested with the same pair of restriction enzymes. The ligation of the DNA insert was conducted overnight at 16℃ using T4 DNA ligase. E. coli BL21(DE3) cells were transformed with the ligation mixture and plated on Luria–Bertani (LB) agar containing kanamycin(50 μg/ml). Positive colonies were screened by direct colony PCR using vector-specific primers (T7 promoter and T7 terminator primers). E. coli BL21(DE3) cells, transformed with the recombinant plasmid pET-30a, were grown in LB medium containing 50 μg/ml kanamycin on a rotary shaker at 200 rpm at 37oC. When the absorbance at 600 nm reached 0.6, 0.1 mM IPTG was added to the culture medium, and the cultures were incubated further at 25℃ for 12 h. Cells were harvested by centrifugation at 8,000 rpm for 10 min at 4oC. Based on the protocol of the Ni-NTA His-Bind Purification kit, the cell pellet was lysed in 50 mM phosphate buffer (pH 8.0) by sonication, followed by centrifugation at 12,000 rpm for 30 min. The clarified crude lysate was loaded onto a 7 ml Ni–NTA column after adding 300 mM NaCl and 20 mM imidazole. After binding, the column was washed with 1 column-volume of buffer A (50 mM phosphate buffer, 300 mM NaCl, and 20 mM imidazole), followed by 5 column-volumes of buffer B (50 mM phosphate buffer, 300 mM NaCl, and 50 mM imidazole). The bound enzyme was eluted with buffer C (50 mM phosphate buffer, 300 mM NaCl, and 200 mM imidazole) and the active fractions were pooled, desalted, and concentrated for further analysis. Protein concentration was determined by the method of Bradford [1] using bovine serum albumin as the standard. β-Galactosidase activity was determined using ONPG as the substrate. A 0.5 ml aliquot of enzyme solution was added to 2 ml of ONPG solution and incubated for 15 min. The reaction was stopped by the addition of 0.5 ml of 10% sodium carbonate, and the absorbance was determined at 420 nm. One β-galactosidase unit was defined as the quantity of enzyme that would liberate 1 μM of o -nitrophenol (ONP) from ONPG per minute under the assay conditions. The molecular mass of the subunit was estimated by SDS-PAGE, using a vertical gel electrophoresis system. SDS-PAGE was performed according to the procedure described by Laemmli [10] . The β-galactosidase activity was determined in different pH buffers (sodium acetate (pH 2.0-5.0), sodium phosphate (pH 5.0-8.0), and Tris–HCl (pH 8.0-10.0)). Enzyme solution and substrate (ONPG) were mixed with the buffer solution and incubated at 45℃ for 15 min. The effect of temperature on β-galactosidase activity was determined at different temperatures ranging from 20℃ to 70℃. The activity of the above treated samples was assayed by the standard method, as given above. The relative activity was expressed as the ratio of β-galactosidase activity under a certain condition to its maximum activity. The kinetic constants (V _max and K _m ) were determined using Lineweaver–Burk double reciprocal (1/V versus 1/S) plots, where substrate ONPG concentrations of 0.33-2 mM at pH 7.0 and 37℃ were used. The effects of metal ions (K ⁺ , Ca ²⁺ , Mg ²⁺ , Mn ²⁺ , Zn ²⁺ , and Cu ²⁺ ) on β-galactosidase activity were evaluated using sodium phosphate buffer (pH 7.0). The enzyme was incubated for 30 min at 37℃ with 5 mM of metal ions prior to the addition of substrate. The activity without added metal ions was taken as 100% activity. In order to determine the thermostability of the crude enzyme, residual activities were measured after incubating the enzyme at 50℃ from 2 to 120 h. Three genes designated lacLM, lacL , and lacM were cloned from L. kefiranofaciens ZW3, which encode a putative heterodimeric LacLM-type β-galactosidase and its large and small subunits, respectively. lacLM (2,833 bp) encodes a protein of 948 amino acids with a calculated molecular mass of 110 kDa and is composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp), that encode large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. Moreover, the lacM gene is found downstream of lacL , and the two genes overlap for 17 bp ( Fig. 1 ). Based on the carbohydrate-active enzymes databank ( http://www.cazy.org ) and amino acid sequence alignment, β-galactosidase from L. kefiranofaciens ZW3 can be classified as a member of the GH2 family. Comparison of the amino acid sequences of β-galactosidase with those from other Lactobacillus GH2 members is shown in Fig. 2 . Inspection of the amino acid sequence alignment revealed that the catalytic residues of β-galactosidase from L. kefiranofaciens ZW3 are Glu415, His417, and Glu536 of LacL, corresponding to Glu416, His418, and Glu536 of LacL from L. reuteri L103 [15] . As expected, these amino acid residues are highly conserved in all GH2 members. To further study the LacLM-type β-galactosidase character of L. kefiranofaciens ZW3, the lacLM, lacL , and lacM genes were cloned into pET-30a and expressed successfully in E. coli BL21(DE3) ( Fig. 3 ). Each protein was expressed as a soluble protein containing a hexa-histidine tag at both the N- and C-terminus. Then, LacLM, LacL, and LacM were purified on a Ni-NTA column ( Fig. 3 ). The activities of recombinant β-galactosidase ( i.e. , holoenzyme), recombinant large subunit, and small subunit for hydrolyzing ONPG were investigated. In addition, in order to study the relationship between the two subunits of β-galactosidase, the two recombinant subunits were mixed at a 50:50 ratio in vitro. Table 2 shows that the recombinant large subunit had activity besides the recombinant holoenzyme, which has never before been reported. The studies of Nguyen et al. [17] showed that the recombinant large subunit of β-galactosidase from L. reuteri L103 does not have any activity. The opposite result might partly be due to the difference in the amino acid sequence of the large subunit of β-galactosidase, since those of L. kefiranofaciens ZW3 and L. reuteri L103 share only 75.8% identity. Furthermore, the K _m value of the holoenzyme was 2.3-times higher than that of the large subunit, which indicates that the large subunit has a higher affinity for ONPG than does the holoenzyme. On the contrary, the V _max value of the holoenzyme for ONPG was 8.9-times higher than that of the large subunit, which means that the activity of the holoenzyme was higher than that of the large subunit. Moreover, the V _max value of the mixture of the two subunits was also 1.7-times higher than that of the single large subunit. However, the recombinant small subunit expressed by the lacM gene displayed no hydrolytic activity. However, it improved the V _max of the large subunit for ONPG after being mixed with the latter in vitro. These results suggest that the β-galactosidase catalytic center is mainly located in the large subunit, and after being translated in vivo, two subunits immediately combine to form a more efficient and stable structure for activity. However, when the two subunits were mixed in vitro, a part of the subunit might denature, considering environmental conditions. Alternatively, the structure of each subunit might become unstable as the large or small subunit alone existed for a long time before being mixed. All of these factors could reduce their binding rate, which would negatively influence formation of the holoenzyme. Consequently, the V _max of the mixture of the two subunits in vitro was lower than that in vivo. Further work will be needed to confirm the structural features of the two subunits and how they bind with each other. The effects of pH and temperature on β-galactosidase activity were examined as shown in Figs. 4 A and 4 B. The results indicate that there was no difference between the large subunit and large-small subunit mixture in terms of hydrolytic activity for ONPG under different pH and temperature conditions ( Figs. 4 A and 4 B). Moreover, the optimum temperature and pH of the holoenzyme and large subunit are 60℃ and 8.0, and 50℃ and 7.0, respectively. In addition, the hydrolytic activity of the large subunit and the holoenzyme remained at 80% when the temperature was decreased to 40℃ and 48℃, respectively. Furthermore, the hydrolytic activity showed a rather narrow optimum (pH 6.0-8.5) in both the large subunit and large-small subunit mixture, whereas a broader optimum (pH 5.0–8.5) was obtained for the holoenzyme. Similarly, the suitable pH of hydrolytic activity in the large subunit and largesmall subunit mixture was 5.0-9.0, and 4.0-9.0 in the holoenzyme, since the recombinant proteins could retain more than 50% of the maximum activity. The effects of ions on β-galactosidase activity were analyzed. As shown in Table 3 , Zn ²⁺ , Ca ²⁺ , Cu ²⁺ , and Mn ²⁺ had negative effects on the activity of the recombinant proteins, whereas Mg ²⁺ exhibited a positive effect. Moreover, K ⁺ had slight influence on β-galactosidase hydrolytic activity. These results are consistent with those of previous reports [4 , 7 , 21] . However, the sensitivity of the holoenzyme to negative regulatory ions was higher than that of the other recombinant proteins. Thus, it was suggested that these negative regulatory ions might partly break down the combination of the two subunits of the holoenzyme. As a result, holoenzyme activity dropped more than that of the recombinant large subunit. The hydrolytic activity of the three recombinant proteins decreased with increasing incubation time at 50℃ and all of them reached their lowest activity after incubation for 120 h ( Fig. 5 ). Interestingly, compared with the large subunit, the mixture of the recombinant large and small subunits displayed obviously higher thermostability, whereas the thermostability was lower than that of the holoenzyme. These results indicate that the small subunit plays an important role in maintaining the stability of the structure of β-galactosidases. In conclusion, one LacLM-type β-galactosidase gene was cloned from L. kefiranofaciens ZW3, which was isolated from kefir grains. The gene is composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding the large and small subunits of β-galactosidase, respectively. The three genes were successfully expressed in E. coli DE3 and recombinant proteins were purified and characterized. Compared with the holoenzyme of β-galactosidase encoded by the lacLM gene, the recombinant large subunit exhibited obviously lower thermostability and hydrolytic activity for ONPG. As for the holoenzyme and large subunit, they showed maximum ONPG hydrolysis activity at 60℃ and pH 8.0, and 50℃ and pH 7.0, respectively. Although the recombinant small subunit alone had no ability to hydrolyze ONPG, it could improve the hydrolytic activity and thermostability of the recombinant large subunit after its addition to the latter in vitro. Therefore, the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.