title: 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).
  
  
  CONCLUSIONS
  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.
authors.forename: Xia
authors.initials: X
authors.lastname: Zhang
authors.forename: He
authors.initials: H
authors.lastname: Li
authors.forename: Chang-Jie
authors.initials: CJ
authors.lastname: Li
authors.forename: Teng
authors.initials: T
authors.lastname: Ma
authors.affiliation: School of life sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China. lsslig@mail.sysu.edu.cn.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.forename: Yu-Huan
authors.initials: YH
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D002681:China; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D005947:Glucose / Q000378:metabolism*; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D005947:Glucose; D005690:Galactose