scholarly journals New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 α-amylase contributes to starch binding and raw starch degrading

2000 ◽  
Vol 350 (2) ◽  
pp. 477-484 ◽  
Author(s):  
Jun-ichi SUMITANI ◽  
Tadashi TOTTORI ◽  
Takashi KAWAGUCHI ◽  
Motoo ARAI
Keyword(s):  
Molecular Mass ◽  
Catalytic Domain ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
Binding Domain ◽  
Terminal Region ◽  
Proteolytic Processing ◽  
Bacillus Sp ◽  
Raw Starch ◽  
Raw Starch Binding

The α-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to α-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pHand thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69kDa, form (BAA-α) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60kDa, form (BAA-β), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-α. The smallest, 50kDa, form (BAA-γ) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-α was specifically adsorbed on starch or dextran (α-1,4 or α-1,6glucan), and specifically desorbed with maltose or β-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

scholarly journals Introduction of Raw Starch-Binding Domains intoBacillus subtilis α-Amylase by Fusion with the Starch-Binding Domain of Bacillus Cyclomaltodextrin Glucanotransferase

2000 ◽  
Vol 66 (7) ◽  
pp. 3058-3064 ◽  
Author(s):  
Kohji Ohdan ◽  
Takashi Kuriki ◽  
Hiroki Takata ◽  
Hiroki Kaneko ◽  
Shigetaka Okada
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Raw Starch ◽  
Chimeric Enzymes ◽  
Binding Domains ◽  
Molar Basis ◽  
Effects Of Ph ◽  
Raw Starch Binding ◽  
Cyclomaltodextrin Glucanotransferase

ABSTRACT We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of Bacillus subtilis X-23 α-amylase (Ba-S) and the raw starch-binding domain (domain E) of Bacillus A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT). Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT. Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S). Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis. Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S. We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy.

scholarly journals Raw-starch-digesting and thermostable α-amylase from the yeast Cryptococcus sp. S-2: purification, characterization, cloning and sequencing

1996 ◽  
Vol 318 (3) ◽  
pp. 989-996 ◽  
Author(s):  
Haruyuki IEFUJI ◽  
Mariko CHINO ◽  
Miyoshi KATO ◽  
Yuzuru IIMURA
Keyword(s):  
Amino Acids ◽  
Terminal Region ◽  
Cloning And Sequencing ◽  
Raw Starch ◽  
Amylolytic Enzymes ◽  
Binding Motifs ◽  
Reading Frame ◽  
Putative Signal Peptide ◽  
Raw Starch Binding ◽  
One Step

A starch-degrading enzyme produced by the yeast Cryptococcus sp. S-2 was purified in only one step by using an α-cyclodextrin–Sepharose 6B column, and was characterized as an α-amylase (EC 3.2.1.1). The molecular mass and isoelectric point of purified α-amylase (AMY-CS2) were estimated to be 66 kDa and 4.2 respectively. AMY-CS2 has raw-starch-digesting and raw-starch-absorbing activities. Furthermore it was shown to be thermostable. An open reading frame of the cDNA specified 611 amino acids, including a putative signal peptide of 20 amino acids. The N-terminal region of AMY-CS2 (from the N-terminus to position 496) had 49.7% similarity with the whole region of α-amylase from Aspergillus oryzae (Taka-amylase), whereas the C-terminal region had a sequence that was similar to the C-terminal region of glucoamylase G1 from A. niger. In addition, putative raw-starch-binding motifs exist in some amylolytic enzymes. A mutant AMY-CS2 that lacks the C-terminal domain lost not only its ability to bind or digest raw starch, but also its thermostability. Consequently it is possible that the putative raw-starch-binding domain of AMY-CS2 plays a role not only in the molecule's raw-starch-digesting ability but also in its thermostability.

scholarly journals Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain

FEBS Journal ◽  
2006 ◽  
Vol 273 (10) ◽  
pp. 2161-2171 ◽  
Author(s):  
Jozef Sevcik ◽  
Eva Hostinova ◽  
Adriana Solovicova ◽  
Juraj Gasperik ◽  
Zbigniew Dauter ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Domain ◽  
Raw Starch ◽  
Raw Starch Binding

scholarly journals An α-l-Arabinofuranosidase fromTrichoderma reesei Containing a Noncatalytic Xylan-Binding Domain

1999 ◽  
Vol 65 (9) ◽  
pp. 3964-3968 ◽  
Author(s):  
Masahiro Nogawa ◽  
Kenji Yatsui ◽  
Akiko Tomioka ◽  
Hirofumi Okada ◽  
Yasushi Morikawa
Keyword(s):  
Molecular Mass ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Crystalline Cellulose ◽  
Terminal Amino Acid ◽  
Catalytic Constant ◽  
Terminal Amino ◽  
Oat Spelt

ABSTRACT l-Sorbose, an excellent cellulase and xylanase inducer from Trichoderma reesei PC-3-7, also induced α-l-arabinofuranosidase (α-AF) activity. An α-AF induced by l-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported α-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported α-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters forp-nitrophenyl-α-l-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported α-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.

scholarly journals Molecular and Biochemical Characterization of Two Xylanase-Encoding Genes from Cellulomonas pachnodae

1999 ◽  
Vol 65 (9) ◽  
pp. 4099-4107 ◽  
Author(s):  
Anne E. Cazemier ◽  
Jan C. Verdoes ◽  
Albert J. J. van Ooyen ◽  
Huub J. M. Op den Camp
Keyword(s):  
Amino Acids ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Binding Domain ◽  
Cellulolytic Bacterium ◽  
Glycosyl Hydrolases ◽  
Binding Studies ◽  
Calculated Molecular Mass ◽  
Encoding Genes

ABSTRACT Two xylanase-encoding genes, named xyn11A andxyn10B, were isolated from a genomic library ofCellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the twoCellulomonas species were identified.

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