Introduction galactosidase d galactoside galactohydrolase

Introduction β-galactosidase (β-d-galactoside galactohydrolase or lactase; EC 3.2.1.23) is an important type of glycoside hydrolase that can catalyze the conversion of lactose to p-selectin and galactose. Furthermore, it can also catalyze transglycosylation reactions, which are usually used for the synthesis of galactooligosaccharides (GOSs) [1]. During the last decade, β-galactosidases have drawn considerable interest due to two main biotechnological applications: removal of lactose from milk for lactose-intolerant people [2], and the production of GOSs as attractive prebiotics [3]. Additionally, β-galactosidase is commonly used in the dairy industry to improve the quality, nutrition, and taste of food products due its ability to carry out the enzymatic hydrolysis of lactose [4]. Moreover, its transgalactosylation activity is widely used to synthesize galactosylated products [5]. Microbial β-galactosidases have drawn particular attention because of their high yields, high activity, and abundance [6,7]. In nature, only 0.1–1% of the bacterial species have been examined to be cultivable by traditional methods [6]. In recent years, with the development of sequencing technology, screening unculturable microorganisms by sequence-based alignment to known sequences in the metagenomic database has been a useful approach for identifying novel β-galactosidases with desirable properties. β-galactosidases with special features also have wide industrial applications. The thermostable β-galactosidases can increase the yield of GOSs, because high initial concentrations of lactose at higher temperatures can increase the formation of GOS products [8]. Cold-adapted β-galactosidases can produce dairy products at low temperatures, which avoids taste and nutrition changes and reduces the cost of producing lactose-free milk [9]. The alkalophilic β-galactosidases from Meiothermus ruber DMS1279 [10], and Enterobacter cloacae B5 [11] have been researched for their potential use in the biocatalytic production of GOSs. The acidophilic β-galactosidases have been used for producing acidic whey permeate. However, identification of new β-galactosidases with good tolerance and novel features is still required for their improved industrial production. In this study, the screening of novel β-galactosidases from a sequence-based metagenome was carried out. An E. coli β-galactosidase-deficient host was constructed using the CRISPR-Cas9 system, for expressing selected β-galactosidase genes. From thirty selected β-galactosidases, twelve clones showed β-galactosidase activity and were examined to research the temperature and pH conditions required for their optimal activity. Finally, four β-galactosidases with good features were purified for further study. Using this efficient technique for mining new β-galactosidases from the sequence-based metagenome, we identified some novel β-galactosidases with potential for industrial applications.
Materials and methods
Results
Discussion Some microbial β-galactosidases with higher activities and good features have been reported previously [9,10,14]. Screening from metagenomic libraries is an appropriate method for discovering novel β-galactosidases. E. coli BL21(DE3) is one of the most efficient prokaryotic host strains; it has been widely used for expressing enzymes [15]. However, the β-galactosidase gene, lacZ, is present in E. coli BL21(DE3) competent cells [16]. Therefore, the E. coli DH5α [17] and JM109 cells [18] were used as an alternative for expressing β-galactosidase genes. Our host E. coli ΔlacZ(DE3)pRARE was derived from E. coli BL21(DE3), which is more efficient for heterologous expression than the DH5α and JM109 strains. BL21(DE3) is a protease-deficient strain, and thus, proteins will not be degraded. The total knockout of the β-galactosidase gene, lacZ, via the CRISPR-Cas9 system, and the insertion of the pRARE plasmid, a helper plasmid encoding tRNA genes for rare codons, into the ΔlacZ(DE3) cells ensured the efficient expression of the exogenous β-galactosidase genes.