Cloning, Expression And Functional Analysis Of Two β-Galactosidases From The Lac Gene Cluster In Lactobacillus Acidophilus

β-Galactosidases (EC 3.2.1.23) that are widely distributed in nature hydrolyse theβ-1,4-D-galactosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety, such as lactose, o-nitrophenyl-β-D-galactopyranoside (ONPG), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and so on.β-Galactosidases were divided into four glycoside hydrolase families: GHF-1, GHF-2, GHF-35 and GHF-42 on the basis of DNA or deduced amino acid similarity. For application in dairy industry and molecular biology, much attention has been focused on the enzymes. Many bacteria have a lactose (lac) operon which contains a GHF-2β-galactosidase gene and other genes involved in lactose utilization, such as Escherichia coli, Lactobacillus delbrueckii. Even though the microorganisms isolated from lactose-rich environment have not only one familyβ-galactosidase, their lactose operons also contain a single GHF-2β-galactosidase gene. And otherβ-galactosidase genes locate near other glycoside hydrolase genes at else site of genome and involve in utilization of complicated polysaccharides.Lactobacillus acidophilus is generally regarded as safe (GRAS). As probiotics and a member of normal flora, it is often used to maintain and enhance health of people and always is one of hot spots of probiotics study. Recently, the sequencing of L. acidophilus NCFM whole genome(GenBank accession no. CP000033) was completed. On the genome, there is a lac gene cluster that contains threeβ-galactosidase genes that encode twoβ-galactosidases belonging respectively to GHF-42 (LacZ) and GHF-2 (LacLM), and other genes involved in lactose metabolism. It is interesting to study why the lac gene cluster of L. acidophilus contains both GHF-42β-galactosidase gene and GHF-2β-galactosidase gene other than the lac operon of E.coli.The lacL and lacM of the lac gene cluster in L. acidophilus co-encode a heterodimeric GHF-2β-galactosidase. The earlier foundβ-galactosidases are all encoded by a single gene. Up to recent years, the heterodimericβ-galactosidase encoded by two genes was found one after another. Nguyen et al. purified the LacLM from L. acidophilus R22 and reported some enzymatic properties of it, but the LacLM still was not cloned and overexpressed. Up to now, there has no report about functional analysis of the LacZ. W.M.Russell et.al described that L. acidophilus lost all activity ofβ-galactosidase when an integrated plasmid inserted into the lacL gene, even though the lacZ gene was not destroyed. The result suggests the lacZ gene of L. acidophilus is inactive. But the other study found transcription level of lacZ, lacL and lacM genes were all significant up in the presence of lactose. The result suggests the lacZ gene can be induced by lactose and may be functionally active. So whether the lacZ gene of L. acidophilus is functional gene remains unknown. What role the lacZ gene does play in lactose utilization, as one ofβ-galactosidase of the lac gene cluster, if the lacZ gene is a functional one?In order to characterize function of the lacZ and study application of the LacZ and the LacLM to dairy industry, following work had been done.1. Cloning, expression and purification of the LacZβ-galactosidase in Escherichia coli. The lacZ gene of Lactobacillus acidophilus encodes a glycoside hydrolase family 42β–galactosidase (76, 582 Da). In our study, the lacZ gene was cloned into pQE31 and pET22b from L. acidophilus ATCC 4356 and the recombinant plasmids were transformed into competent E. coli strain JM109 or Rosetta. Our data showed that recombinant protein without His-Tag hasβ-galactosidase activity. The His-tagged LacZ was expressed as inactive inclusion bodies, and their activity did not recovered by dialysis renaturation. The specific activity ofβ-galactosidase (ONPG) of crude extract from the optimally induced culture was 1.14 U/mg protein. The homogenous enzyme was obtained and has a specific activity of 43.2 U/mg protein after purification by ammonium sulfate fractionation, affinity chromatography, anion-exchange and gel permeation.2. Cloning, expression and purification of the LacLMβ-galactosidase in E. coli The lacL and lacM genes of the sequencing strain NCFM encode polypeptides with calculated molecular masses of 73,253 and 35,817 Da, respectively. The genes were cloned into pQE31 from L. acidophilus ATCC 4356 and were functionally expressed in E. coli JM109. Our data showed amino acid residue 512 of LacL was different from that of known LacL of Lactobacillus acidophilus strains. The specific activity (ONPG) of crude extract from the optimally induced culture was 1.30 U/mg protein. The homogenous recombinant LacLM was obtained and has a specific activity of 226 U/mg protein after purification by ammonium sulfate fractionation, anion-exchange, affinity chromatography and gel permeation.3. Characterization of induced expression ofβ-galactosidases in L. acidophilus ATCC 4356. After rude purification by ammonium sulfate fractionation, the sonicated extract from induced culture of L. acidophilus ATCC 4356 was assayed by native-PAGE, which markers were the recombinant LacZ and LacLM. Then the gel was stained by active solid-state staining. The results showed that the native LacZ and LacLM do exist in induced L. acidophilus ATCC 4356.4. The recombinant enzymes assay. The results of gel permeation and enzyme assay showed that the recombinant LacZ has a native molecular weight of 71.6 kDa, an optimum temperature of 36～40℃, an optimum pH of 6-6.6, a Km value for ONPG of 3.84±0.09 mmol/L and a Vmax value of 52.1±0.6 U/mg protein, and the recombinant LacLM has a native molecular weight of 96.3 kDa, an optimum temperature of 49℃, an optimum pH of 7, a Km value for ONPG of 2.18±0.12 mmol/L and a Vmax value of 273±5 U/mg protein. Especially, the recombinant LacZ keep 16.5 % activity at 4℃, and the recombinant LacLM almost keep 50 % activity at 60℃.The results of cloning and expression of the lacZ gene suggest the lacZ gene is a real gene which encoding protein hasβ-galactosidase activity. Then we testified that the native LacZ does exist in parent strain by active solid-state staining. So the lacZ gene of the lac gene cluster is a real gene and can be normally expressed in parent strain. It suggests the LacZ is involved in lactose utilization.The marked difference of enzymatic properties between recombinant LacZ and recombinant LacLM is the range of temperature. The optimum temperature of recombinant LacZ is 36～40℃, and the enzyme retains 16.5 % activity at 4℃but inactivates at 55℃. However, The optimum temperature of recombinant LacLM is 49℃, and the enzyme retains 50 % activity at 60℃but inactivates at 4℃. So we infer that L. acidophilus uses lactose by LacZ when the strain lives in cold environment (refrigerator, sauerkraut jar), using lactose by LacZ and LacLM in warm environment (intestinal tract) and using lactose by LacLM in hot environment (fermenter, cheese). The lac gene cluster of L. acidophilus contains both GHF-2 and GHF-42β-galactosidases other than the lac operon of E.coli containing a single GHF-2β-galactosidase. The lac gene cluster containing the twoβ-galactosidases makes L. acidophilus use lactose at a more large range of temperature than E.coli. Especially, the strain can use lactose at 4℃.The dairy products must be disinfection by heating and cold preservation in the process of production. If adding recombinant LacZ in the process of cold preservation, or adding recombinant LacLM in the process of disinfection, the lactose-free milk would be obtained. Moreover, there has not safe problem because the enzymes come from a GRAS bacterium. So the enzymes may be useful in production of lactose-free milk for people suffering from lactose intolerance. The hetero-expression yields of recombinant LacZ and LacLM was 5 times more than their yields of parent strain and a possible purified proposal was provided for industry. This study makes base for application of recombinant LacZ and LacLM.