Glucoamylase

Glycogen was analysed according to a modified method as described by Aklujkar et al. [44 (link)]. To 30 mg of dry weight cells 500 μl of 2 M NaOH was added followed by boiling for 1 h. To neutralize this mixture, 60 μl of 9 M H₂SO₄ and 600 μl 1 M NaAc/HAc buffer (pH 4,5) were added. To 400 μl of sample, 50 units of glucoamylase (Novozymes art. no. NS22035) were added and incubated at 50 °C for 1 h. The final glucose concentration of the mixture was measured with and without amyloglucosidase treatment on a Cobas Mira Plus using the Horiba ABX Pentra Glucose HK CP reagent (art. no. A11A01667). Maltose was used as a positive control to check the activity of the amyloglucosidase.

Acetoin (>98%) was purchased from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). Acetonitrile (HPLC grade) was obtained from Sinopharm Chemical Reagent (Shanghai, China). Cassava powder and cottonseed meal were respectively obtained from Guangxi State Farms Mingyang Biochemical Group, Inc. (Nanning, China) and Jinan Oumi Biotechnology Co., Ltd. (Jinan, China). Thermostable α-amylase and glucoamylase obtained from Novozymes (Novozymes China Biotechnology Co., Ltd., Tianjin, China) were used for liquefaction and saccharification of raw starch as previously described [7 (link)]. The acid protease was purchased from Imperial Jade Biotechnology Co., Ltd. (Yinchuan, China) for protein hydrolysis [7 (link)].