P nitrophenyl α d galactopyranoside pnp α gal

Rebaudioside D (RD), Rebaudioside M (RM), Rebaudioside A (RA), stevioside, and the crude steviol glycosides extract containing about 45% stevioside were gifts from Zhucheng Haotian Pharm Co., Ltd. (Weifang, China). Rubusoside and steviol were purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, China). Steviolmonoside, steviolbioside, and Rebaudioside B (RB) were prepared by alkaline hydrolysis from rubusoside, stevioside, and RA, respectively, with 0.5 N NaOH at 100°C for 15 min in this work (Nakano et al., 1998 (link)). p-Nitrophenyl-β-D-glucopyranoside (pNP-β-Glc), p-nitrophenyl-N-acetyl-β-D-glucosamine (pNP-β-Glc NAc), p-nitrophenyl-β-D-glucuronic acid (pNP-β-GlcA), p-nitrophenyl-β-D-galactopyranoside (pNP-β-Gal), p-nitro -phenyl-β-fucose (pNP-β-Fuc), p-nitrophenyl-β-manno -pyranoside (pNP-β-Man), p-nitrophenyl-α-D-glucopyranoside (pNP-α-Glc), and p-nitrophenyl-α-D-galactopyranoside (pNP-α-Gal) were purchased from Sigma-Aldrich (United States). Esculin, cellobiose, laminaribiose, and gentiobiose were purchased from J&K Scientific Co., Ltd. (Beijing, China). The Phusion DNA polymerase was KOD-Plus-Neo from Toyobo Co., Ltd., Life Science Department (Osaka, Japan). T5 Exonuclease and restriction enzymes were from New England Bio-Labs. Glucose Assay Kit was from Biosino Bio-Technology and Science Inc. (Beijing, China). The other chemicals used were analytical grade.

The restriction and modification enzymes in this work were obtained from New England Biolabs (Beijing, China). P-nitrophenyl α-D-galactopyranoside (PNP-α-Gal), yeast nitrogen base without amino acids, agar, PEG3350, dimethyl sulfoxide (DMSO), lithium acetate, and glucose were all purchased from Sigma (Shanghai, China). The yeast expression plasmids pGADT7 and pGBKT7 were from Clontech (Palo Alto, CA), and CDCA was from Merck. The dropout supplement free from leucine and tryptophan (-Leu/-Trp DO supplement) was bought from Takara, and Salmon Sperm DNA was obtained from invitrogen. The yeast strain AH109 was purchased from Clontech (Palo Alto, CA).