E coli trans5α

The strains and plasmids used in this study were listed in additional file (Additional file 1: Table S1). E. coli Trans5α (TransGen, Beijing, China) was used for plasmid construction. E. coli BL21(DE3) and E. coli BL21(DE3)pLysS (TransGen, Beijing, China) were used as the hosts for Bgl1A(A24S/F297Y) production. Ampicillin, chloramphenicol, and IPTG were purchased from Sangon Biotech (Shanghai, China). p-Nitrophenyl β-D-glucopyranoside (pNPG) was from Sigma-Aldrich (St. Louis, MO, USA). The insoluble microcrystalline cellulose with an average particle size of 25 μm was acquired from Aladdin Chemistry (Shanghai, China). Ni²⁺-charged chelating sepharose fast flow was purchased from GE Healthcare (Uppsala, Sweden). All other chemicals were of analytical grade unless otherwise specified. Standard TB medium (per liter contains 4 g glycerol, 24 g yeast extract, 12 g peptone, 17 mM KH₂PO₄, and 72 mM K₂HPO₄) was used as culture medium in 250-mL Erlenmeyer flasks. Modified TB medium (per liter contains 15 g glycerol) was employed in high cell density culture (HCDC), the feeding solutions contained 45 g tryptone, 45 g yeast extract, and 500 g glycerol per liter.

Ni-NTA agarose resin was purchased from Qiagen (Hilden, Germany). Trans5K DNA marker, T4 DNA ligase, E. coli Trans5α and BL21(DE3) competent cells were products of TransGen Biotech (Beijing, China). NdeI and XhoI endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). The substrates of pNPA, pNPB, pNPC, pNPL and pNPP were obtained from Aladdin Biotech (Shanghai, China).

pEASY-E1(+) (Transgen, Beijing, China) was used to express the recombinant protein in Escherichia coli BL21(DE3) pLysS (Novagen, Darmstadt, German) as the vector. T4 DNA ligase, E. coli Trans5α, restriction endonucleases, TransFast^® Taq DNA polymerase and isopropyl-β-D-1-thiogalactopyranoside (IPTG) were obtained from Transgen (Beijing, China). p-Nitrophenyl-β-D-glucopyranoside (pNPG), gentiobiose, and cellobiose were obtained from Sigma-Aldrich (Shanghai, China). N-propanol, ethyl acetate and ammonia solution were obtained from Sangon Biotech (Shanghai, China). The DNA extraction kit was purchased from Mega (Mega Co., Ltd., Guangzhou, China). Other chemical reagents used in this study were all analytically pure unless otherwise requested. Luria-Broth comprised yeast extract (5.0 g·L⁻¹), tryptone (10.0 g·L⁻¹), and NaCl (10.0 g·L⁻¹), and the pH was maintained at 7.0, with or without supplementation with agar (15.0 g·L⁻¹).