*2.4. Construction of Expression Plasmids*

*P. galactosidasius* DSM 18751T (BCRC 80657) was cultivated in accordance with the BCRC protocol. The genomic DNA of the bacterium was isolated using the Geno Plus Genomic DNA Extraction Midiprep System (Viogene) according to the manufacturer's protocol. The target gene (*Pg*MA) was amplified from the genomic DNA with polymerase chain reaction (PCR). The primer set used in the PCR was as follows: forward 5 -gggggatccgttgaaagaagccatttatcatcg-3 and reverse 5 -gggctcgagtcaattttctacttgatagaggag-3 , which contain BamHI and XhoI restriction sites (underlined mark) for cloning. The amplified DNA fragment (1.8 kb length) was cloned into the expression plasmid pET-Duet-1, named pETDuet-*Pg*MA, which was then transformed into *E. coli* BL21 (DE3) for the recombinant *Pg*MA.

#### *2.5. Production and Purification of Recombinant PgMA in E. coli*

The recombinant *E. coli* harboring the recombinant expression plasmid pETDuet-*Pg*MA was cultivated in Luria–Bertani (LB) medium containing 1% (*w*/*v*) tryptone and sodium chloride and 0.5% (*w*/*v*) yeast extract to the optical density at 560 nm (OD560) of 0.6 and then induced with 0.2 mM of IPTG. After further cultivation at 18 ◦C for 20 h, the cells were centrifuged at 4500× *g* and 4 ◦C for 20 min. The cell pellet was washed and spun down twice with 50 mM of phosphate buffer (PB) at pH 6.8 and then broken with sonication via a Branson S-450D Sonifier (Branson Ultrasonic Corp., Danbury, CT, USA). The sonication program was run for five cycles of 5 s on and 30 s off at 4 ◦C. The mixture was then centrifuged at 15,000× *g* and 4 ◦C for 20 min to remove the cell debris. A supernatant containing the recombinant *Pg*MA fused with a His-tag in its N-terminal was applied in an Ni2+ affinity column. The His-tag-fused *Pg*MA was washed with PB with 25 mM imidazole and eluted with PB containing 250 mM imidazole. The eluate was then concentrated and desalted through Macrosep 10 K centrifugal filters (Pall, Ann Arbor, MI, USA). The concentration of the purified *Pg*MA was determined using the Bradford method [39] and analyzed with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The purified *Pg*MA was stored in a final concentration of 50% glycerol at −80 ◦C before use.
