The complete amino acid sequence of taka-amylase A.

Introduction.Taka-amylase A (TAA) [EC 3.2.1.1 a-1,4-glucan 4-glucanohydrolase,Aspergillus oryzae] which was crystallized first by Akabori et al. in 19511) is a glycoprotein consisting of a single polypeptide chain of 478 amino acid residues with an amino (N)terminal alanine and a carboxy (C) -terminal serine.2~'3>The partial amino acid sequences of the N-and C-terminal regions,4~ '5> the carbohydrate chain structure° 7) and X-ray crystallographic analysis8> of the enzyme have been reported.This paper describes the complete amino acid sequence of TAA.Materials and methods.Crystalline TAA prepared from Takadiastase Sankyo9> was further purified by DEAE-cellulose column chromatography.10~The homogeneous enzyme judged by polyacrylamide gel electrophoresis was reduced and carboxymethylated.'1The cyanogen bromide cleavage at methionine residues of the reducedcarboxymethylated TAA (RCM-TAA) was performed in 70% formic acid for 24 h at room temperature.After removal of excess reagents by repeated lyophilization, the CNBr fragments were fractionated by gel filtration on a Sephadex G-75 column, by affinity chromatography on a Concanavalin A-Sepharose column, by ion-exchange chromatography on a SP-Sephadex C-25 column or an AG50W x 2 column and by paper electrophoresis.Methionine-containing peptides were isolated from tryptic and chymotryptic digests of maleylated RCM-TAA by a combination of gel filtration on Sephadex G-75 and G-50 columns, paper electrophoresis and high performance liquid chromatography.Amino acid compositions of peptides were determined with a Hitachi 8355 automated amino acid analyzer after hydrolysis of the samples with twice-distilled HCl containing 1 (v/v) phenol in evacuated sealed tubes at 110°C for 24, 48 and 72 h.Sequence analyses of the fragments were mostly performed by automated Edman degradation using a Beckman model 890C liquidphase sequencer in the presence of Polybrenel2) or an LKB 4020