Aspergillus niger β glucosidase

DNA polymerases PrimeSTAR Max (TaKaRa Bio Inc., Shiga, Japan) and KOD-Plus (Ver.2; Toyobo Co., Ltd, Osaka, Japan) were used to amplify mutated DNA. One Shot ^® TOP10 Chemically Competent Escherichia coli (Thermo Fisher Scientific Inc., MA, USA) was used to amplify the plasmid. Pme I (New England Biolabs, MA, USA) was used to linearize the amplified plasmid for the transformation of P. pastoris strain KM71H, which was used for heterologous production of the mutant enzymes. Yeast (BD Biosciences, Miami, USA), peptone (Nihon Pharmaceutical Co., Ltd, Tokyo, Japan), and glycerol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were used for the medium. Phosphoric acid-swollen cellulose (PASC) was prepared as reported. ¹²⁾ Cellulose I _α and III _I were prepared from green algae Cladophora spp. according to the reported method. ¹³⁾Aspergillus niger β-glucosidase was acquired from Megazyme Ltd. (Wicklow, Ireland).

Culture supernatants of WT or mutant Pc Cel6A (10 µL) were incubated with 100 µg of PASC or cellulose III _I prepared as described previously ¹²⁾¹³⁾ in 100 mM sodium acetate buffer (pH 5.0) with a total volume of 200 µL using 96-well plates at 50 or 60 °C with shaking at 1,000 rpm. After two hours of incubation, the solutions were filtered using 96-well plates with a 0.22 µm filter (MultiScreen ^® Filter Plates, Merck Millipore, MA, USA). Then, 5 µL of 40 U/mL Aspergillus niger β-glucosidase (Megazyme Ltd.) was added to 100 µL of filtrate, and the plates were incubated at 60 °C for 48 h with shaking at 1,000 rpm. After hydrolysis, the solutions were heated at 98 °C for 3 min and filtered again. The concentration of glucose in filtrate was quantified using Glucose CII-Test Wako (FUJIFILM Wako Pure Chemical Corporation) by measuring the absorbance at 492 nm with a Thermo Scientific Multiscan ^® FC (Thermo Fisher Scientific).