Metagenomic approach for the isolation of a thermostable β-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin

Abstract

Background: β-Galactosidases can be used to produce low-lactose milk and dairy products for lactose intolerant people. Although commercial β-galactosidases have outstanding lactose hydrolysis ability, their thermostability is low, and reaction products have strong inhibition to these enzymes. In addition, the β-galactosidases possessing simultaneously high thermostability and tolerance of galactose and glucose are still seldom reported until now. Therefore, identification of novel β-galactosidases with high thermostability and tolerance to reaction products from unculturable microorganisms accounting for over 99% of microorganisms in the environment via metagenomic strategy is still urgently in demand.

Results: In the present study, a novel β-galactosidase (Gal308) consisting of 658 amino acids was identified from a metagenomic library from soil samples of Turpan Basin in China by functional screening. After being overexpressed in Escherichia coli and purified to homogeneity, the enzymatic properties of Gal308 with N-terminal fusion tag were investigated. The recombinant enzyme displayed a pH optimum of 6.8 and a temperature optimum of 78 °C, and was considerably stable in the temperature range of 40 °C - 70 °C with almost unchangeable activity after incubation for 60 min. Furthermore, Gal308 displayed a very high tolerance of galactose and glucose, with the highest inhibition constant K(i,gal) (238 mM) and K(i,glu) (1725 mM) among β-galactosidases. In addition, Gal308 also exhibited high enzymatic activity for its synthetic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG, 185 U/mg) and natural substrate lactose (47.6 U/mg).

Conclusion: This study will enrich the source of β-galactosidases, and attract some attentions to β-galactosidases from extreme habitats and metagenomic library. Furthermore, the recombinant Gal308 fused with 156 amino acids exhibits many novel properties including high activity and thermostability at high temperatures, the pH optimum of 6.8, high enzyme activity for lactose, as well as high tolerance of galactose and glucose. These properties make it a good candidate in the production of low-lactose milk and dairy products after further study.

Figure 1

Identification of the active site residues of Gal308 by alignment of the amino acid residues with other five homologous β-galactosidases from GH family 42. The GenBank accession numbers are as follows: Geobacillus thermocatenulatus, AAW56416; Truepera radiovictrix DSM17093, ADI14846; Thermus thermophilus, ABI35985; Alicyclobacillus acidocaldarius, AAZ81841; Bacillus circulans, AAA22260; This study (Gal308), AFD21844. The alignment was carried out using the Clustal W method. The number flanking the sequences represents amino acid positions of each sequence. Asterisks mean identity. The two putative catalytic residues (E195 and E368) of Gal308 were shown in box.

Figure 2

SDS-PAGE analysis of recombinant Gal308 from supernatant of E. coli BL21 (DE3) cell lysates and purified Gal308 by affinity chromatography. Lanes: M, standard protein molecular mass markers (sizes in kilodaltons are indicated on the left); 1, recombinant Gal308 from supernatant of E. coli BL21 (DE3) cell lysates; 2, recombinant Gal308 purified by His•Bind® Purification Kit. The sizes in kilodaltons of protein marker were listed as follows: porcine heart myosin (200,000 Da), E. coli β-galactosidase (116,000 Da), rabbit muscle phosphorylase B (97,200 Da), bovine serum albumin (66,409 Da), ovalbumin (44,287 Da), carbonic anhydrase (29,000 Da).

Figure 3

Effect of pH (A) and temperature (B) on activity (square) and stability(circle) of Gal308 using lactose as substrate. Data points are the average of triplicate measurements; error bars represent ±1 SD.

Figure 4

Effects of galactose (circle) and glucose (square) as inhibitors on the activity of Gal308. The reactions were performed under standard conditions with ONPG as a substrate. The relative activity was defined as the relative value to the maximum activity without galactose or glucose. Data represent the means of three experiments and error bars represent standard deviation.