Expression Of BgaB Gene Encoding Thermostable β-galactosidase In Bacillus Subtilis

β-Galactosidase(EC.3.2.1.23), which can hydrolyze β?-1,4-D-galactosidic linkage, isindustrially important for their applications to produce lactose-free milk. It is necessary to use athermostable enzyme, capable of retaining activity at high temperatures and eliminating thegrowth of methsphilic microflora during operation. In this paper, the gene bgaB encoding thethermostable β-galactosidase from Bacillus stearothermophilus was cloned and expressed inBacillus subtilis.The bgaB gene was constructed into the pZ01 plasmid, which has a constructive promoternamed P43. The recombinant strain showed thermostable β-galactosidase activity of 24U/mLand the target protein band about 70kDa in SDS-PAGE analysis. The study of plasmid stabilityshowed the recombinant strain was stable in antibiotic-free media for 10 days. And afterincubating in 65℃ for 4 hours, the enzyme remained 80% activity, which showed therecombinant enzyme was thermostable in Bacillus subtilis.The promoter and gene sequence were amplified through PCR from the genome of B.stearothermophilus, and constructed into pMK4. SDS-PAGE analysis showed recombinantprotein in BD170/pMK4-bgaB2. The result suggested that the bgaB promoter from B.stearothermophilus was effective in B. subtilis. Then bgaB promoter sequence was constructedinto pZ01-bgaB replacing its own promoter. Compared with former strain WB600/pZ01-bgaBand WB600/pMA5-bgaB(promoter HpaII), bgaB promoter was the weakest.Finally, we tried the secreting expression of bgaB gene. bgaB gene was inserted intosecretion-expression vector pZP01 and pUH, which have signal sequence of Bacillusmegaterium G-Acylase and Bacillus subtilis α-amylase respectively. The two transformantspZP01-bgaB and pUH-bgaB were assayed for enzyme activity, showing that most recombinantprotein stayed within the cell. We regard β?galactosidase itself is an intracellular enzyme andcould not secret effectively in B.subtilis only through signal peptide.