The complete amino acid sequence of taka-amylase A.

Abstract

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.'1 The 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 College,

Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

“…Although there is no general sequence homology between this glucoamylase and known ttamylases (10,18,24,32,33), it was observed that a short stretch of polypeptide chain, preceding Trp(120) in glucoamylase and the Trp(83) in the Taka-amylase A (20) (from A. oryzae) was identical ( Figure 5). The Trp(83) is located in the active site cleft of the three-dimensional structure ofTaka-amylase A and model fitting studies with amylose have recently proposed that Trp(83) participates in binding of this substrate (20).…”

“…100 amino acid residues, bul the two forms have essentially the same activity on soluble substrates (31). The amino acid sequence of the enzyme (5,6,30) near the Abbreviations: AH = aminohexyl; G1 and G2 = the larger and the smaller of the two forms ofglucoamylase from A. niger (31); HPLC = high pressure liquid chromatography; NBS = N-bromosuecinimide; TFA = trifluoroacetic acid; Tris = 2-amin~-2(hydroxymethyl)-l, 3-propandioI. essential residue resembles that near a tryptophanyl residue of Taka-amylase A, proposed to interact with substrate in the active site cleft of this a-amylase (20,33). O.Oz Fine Chemicals, Uppsala, Sweden.…”

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

“…Two positions likely to be glycosylated are Thr (47) and Ser(93). Both were vacant in automated sequencing, but they have tentatively been identified from the amino acid composition of the highly purified and otherwise fully sequenced tryptic fragments T6-4 and T5-2, respectively (43).…”

“…No homology was apparent between the primary structures of Gl and or-amylases described in the literature (14,21,31,37,46,47). Three fungal carbohydrases, a mycodextranase (36), a cellobiohydrolase (9,13), and the glucoamylase G1 (34,44), which all attack insoluble substrates, i.e.…”

Alignments of Tryptic Peptides in CNBr Fragments of α-Amylase fromBacillus subtilisvar.amylosacchariticus

“…7 ) The amino acid sequence of the enzyme from Bacillus amyloliquefaciens has been investigated for a long period of time,S) but only the N-terminal half of the sequence was established. 9 ) In our preceding papers, 1,2) we described that i) the tryptic digest of the rt-amylase generated thirty-four ·peptides, six of which contained methionine residue(s),ii) CNBrcleavage of the enzyme generated eight fragments, …”