Abstract
Backgroundβ-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.ResultsIn 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 Ki,gal (238 mM) and Ki,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).ConclusionThis 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.
Highlights
Background βGalactosidases (EC 3.2.1.23), which hydrolyze lactose to glucose and galactose, have two main applications in food industry, including production of low-lactose milk and dairy products for lactose intolerant people and production of galacto-oligosaccharides from lactose by the transgalactosylation reaction [1]
Screening for β-galactosidase from a metagenomic library To discover novel thermostable β-galactosidases, a metagenomic library consisting of approximately 8,000 clones was constructed using DNA extracted from soil samples of the Mountain of Flames of the Turpan Basin in China
A protein blast search in the databases of NCBI indicated that the protein had the highest identity of 49% (291/599) with the βgalactosidase from one thermophilic microbe Geobacillus thermocatenulatus, as well as a low identity of only 38% (224/593) with the β-galactosidase from the other thermophilic microbe Thermoanaerobacterium thermosaccharolyticum DSM 571, suggesting that Gal308 is probably a novel thermostable β-galactosidase from unculturable microorganisms
Summary
Background βGalactosidases (EC 3.2.1.23), which hydrolyze lactose to glucose and galactose, have two main applications in food industry, including production of low-lactose milk and dairy products for lactose intolerant people and production of galacto-oligosaccharides from lactose by the transgalactosylation reaction [1]. Commercial β-galactosidases are produced from fungi of the genus Aspergillus and yeasts of the genus Kluyveromyces [2] Despite these β-galactosidases have outstanding lactose hydrolysis ability, they have two major drawbacks including low thermostability and high inhibition of reaction products. The optimum termperatures of these enzymes are less than 58°C [3,4], and they have low stability during the high-temperature (65–85°C) pasteurization of milk These enzymes are badly inhibited in the presence of the reaction products (galactose and glucose) [5,6], and the inhibition of reaction products may lead a decrease in the reaction rates or even stop enzymatic reaction completely. Some efforts should be made to discover novel βgalactosidases with high thermostability and tolerance to reaction products from unculturable microorganisms of environment
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
