Cloning and characterization of the Bambusa oldhamii BoMDH-encoded malate dehydrogenase

Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers

“…To determine the kinetic parameters for PAL, a range of L-phenylalanine were used between 0.12 and 12.1 mM. A substrate saturation curve was obtained after a 10 min incubation [33,45]. The K m and k cat values were calculated according to Michaelis-Menten equation [46] and double reciprocal plot [47].…”

NLIP and HAD-like Domains of Pah1 and Lipin 1 Phosphatidate Phosphatases Are Essential for Their Catalytic Activities

“…Removal of disordered regions of Pah1, NLIP and HAD-like domain fusion (1-98-360-591), was expressed in E. coli but could only be detected by anti-His antibodies in the insoluble inclusion body fraction [ 20 ], indicating that hydrophilic disordered regions are required for maintaining Pah1 solubility. Herein, we used another strategy to merge a thioredoxin (Trx) protein at the N-terminus of the fusion protein to increase its protein solubility in Pah1 truncations [ 48 , 49 ]. We also tried other fusion proteins such as maltose-binding protein and glutathione S-transferase; however, thioredoxin fusion protein gave us the best result.…”

“…E. coli DH5α was used for the proliferation of plasmids, and BL21(DE3) was transformed for the expression of the yeast Pah1 and human Lipin 1-α truncations as described previously [ 20 , 49 , 51 ]. E. coli DE3 strains carrying the plasmids ( Table 1 ) were grown at 37 °C in 250 mL of Luria–Bertani (LB, 1% tryptone, 0.5% yeast extract, 1% NaCl) medium containing 100 μg/mL ampicillin.…”

“…E. coli cells were resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, 1 mM phenylmethanesulfonyl fluoride (PMSF), pH 7.4) and disrupted by sonication (Ultrasonic Processors, Sonicator 3000, Misonix, Farmingdale, NY, USA) [ 49 , 51 ]. Cell lysates were added into an open column containing an appropriate amount of Ni-charged resin (Bio-Rad, USA).…”

“…Molecules 2021, 26, 5470 4 of 12 dered regions are required for maintaining Pah1 solubility. Herein, we used another strategy to merge a thioredoxin (Trx) protein at the N-terminus of the fusion protein to increase its protein solubility in Pah1 truncations [48,49]. We also tried other fusion proteins such as maltose-binding protein and glutathione S-transferase; however, thioredoxin fusion protein gave us the best result.…”