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  • Primidone Introduction galactosidase d galactoside galactohy

    2021-09-09

    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 Primidone 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.