pkc inhibitor Many neutral and acidophilic galactosidases ha

Many neutral and acidophilic β-galactosidases have been purified from microorganisms [14], while only a few alkalophilic β-galactosidases have been reported [19]. An alkalophilic β-galactosidase, BGal_375, was obtained and researched for the characterization of its hydrolytic activity. BGal_375 showed a good transglycosylation activity. Two typical types of GOSs, tri- and tetra-saccharides, were obtained. Previous reports have proved that β-galactosidases mainly produce pkc inhibitor (allolactose and 6′-galactobiose) and tri-saccharides (6′-galactosyl lactose) [20,21]. In addition to the tri- and tetra-saccharide peaks, the transglycosylation reaction of β-galactosidase BGal_375 also resulted in an impure peak (with an arrow in Fig. 4A), separate from the lactose peak in HPLC analysis, and unknown spots near the lactose spot in TLC analysis (Supplementary Fig. S4). These were speculated to be representative of the disaccharide allolactose, according to a previous report [20]. However, this needs to be further researched. BGal_375 can be used to synthesize many kinds of galactosyl derivatives, when the galactosyl acceptor is another substance, such as fructose [22], sucrose [23], or n-butanol [24]. BGal_137 is a homodimer, the subunit molecular mass of which is 63 kDa. It is common that microbial β-galactosidases are multimeric proteins with subunit molecular masses mainly between 60 kDa and 80 kDa. The thermostable β-galactosidases from Thermotoga naphthophila RUK-10 [7] are homodimers. BGal_375, BGal_144-3, and BGal_145-2 are all monomeric proteins. The molecular masses of BGal_144-3 and BGal_145-2 were about 60 kDa, which was relatively low compared to the molecular mass of BGal_375, which was 112 kDa. Many monomeric proteins were reported from Thermotoga maritima (78 kDa) [25] and Teratosphaeria acidotherma AIU BGA-1 (180 kDa) [19]. Monomeric proteins are easier to use in industrial applications than multimeric proteins. Relatively low-molecular weight proteins are also normally easier for heterologous expression than high-molecular weight proteins, due to lower resource consumption and quicker folding. Therefore, monomeric β-galactosidases with low molecular weights might serve to produce highly efficient enzymes in a cost-effective manner. In conclusion, an alkaline β-galactosidase efficient for the synthesis of GOSs and three β-galactosidases with good hydrolytic activity were obtained.
Funding This research was supported by the National Natural Science Foundation of China [No. C010302-31500066].
Conflicts of interest
Acknowledgements We thank Prof. Sheng Yang (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) for providing the pTargetF and pCas plasmids.
Introduction β-Galactosidases (EC 3.2.1.23) are enzymes that catalyze the hydrolysis of a glycosidic bond between a terminal non-reducing β-d-galactoside unit and an aglycone moiety (Juers et al., 2012). The term lactase is sometimes used to refer to β-galactosidase, since its main biotechnological application is the manufacturing of lactose-free milk and dairy product; despite that, not all the β-galactosidases are able to hydrolyze lactose (Solomon et al., 2013). Furthermore, β-galactosidases are also able to catalyze transgalactosylation reactions. Briefly, in the first stage the enzyme reacts with a terminal β-galactoside, forming the galactosyl-enzyme complex and releasing the aglycone moiety. In order to release the enzyme active site, in the second stage the galactose moiety is transferred to a water molecule (hydrolysis) or to a different nucleophile containing a hydroxyl-group (transgalactosylation). In the last decade transgalactosylation reactions have gained notoriety since they allow obtaining valuable compounds from lactose, such as: galacto-oligosaccharides (GOS), lactulose, fructosyl-galacto-oligosaccharides (fGOS) and alkyl-galactosydes (Guerrero et al., 2015, Vera et al., 2017). This has driven the search of new β-galactosidases since the synthesis of transgalactosylated compounds is a kinetically controlled reaction, which yield depends mainly on the kinetic properties of the enzyme (Guerrero et al., 2015).