Pseudomonas isoamylase

The distribution of glucan chain length was determined by fluorescence-assisted capillary electrophoresis (FACE) as described previously [47 ]. Starch was solubilized by boiling 4 mg in 200 μl 100% dimethyl sulfoxide. Then 2 μl of the solubilized starch was mixed with 38 μl 500 mM sodium acetate pH 4.4, and debranched by incubating with 2 μL of Pseudomonas isoamylase (Megazyme International. Wicklow, Ireland) at 42°C overnight. Then 10 μl of the debranched starch was dried in Eppendorf Vacufuge vacuum concentrator (Eppendorf, Hauppauge, NY) at 30°C for 1 hour. Then 2 μL of 1 M sodium cyanoborohydride (Sigma-Aldrich Co., St. Louis, MO) and 2 μL of 10% [w/v] 8-amino-1,3,6-pyrenetrisulfonic acid (APTS; Biotium, Inc., Scarborough, ON, Canada) solution made by resuspending 10 mg powdered APTS in 96 μL of 15% [v/v] acetic acid. The samples were separated on a Beckman P/ACE capillary electrophoresis instrument equipped with a laser activated fluorescence detector. The analysis software was used to extract percent peak area as a measure of the relative abundance of glucan chains.