Abstract
Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites.
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Claims 20
Claim · model-written summary
Wild-type barley AMY1 had a multiple attack degree of 1.9.
- Taxon
- Barley
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Hordeum vulgaree0 - Gene
- alpha-amylase 1 (AMY1)e0
- Outcome
- degree of multiple attack (DMA)e0
- Value
- 1.9e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
.jpg?width=500)
Verbatim
e0“Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9”
Claim · model-written summary
Y105A increased multiple attack degree to 3.3.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively”
t0“Barley”
Claim · model-written summary
AMY1-SBD increased multiple attack degree to 3.0.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively”
t0“Barley”
Claim · model-written summary
M53E had decreased DMA in the 1.0-1.4 range.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4”
t0“Barley”
Claim · model-written summary
M298S had decreased DMA in the 1.0-1.4 range.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4”
t0“Barley”
Claim · model-written summary
T212W had decreased DMA in the 1.0-1.4 range.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4”
t0“Barley”
Claim · model-written summary
Y105A/T212W had decreased DMA in the 1.0-1.4 range.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4”
t0“Barley”
Claim · model-written summary
C95A had DMA similar to wild type.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“C95A (subsite -5) had a DMA similar to that of wild type”
t0“Barley”
Claim · model-written summary
Maltoheptaose was the major initial oligosaccharide product.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- Maltoheptaose (G7)e0
- Direction
- majore0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Maltoheptaose (G7) was always the major initial oligosaccharide product”
t0“Barley”
Claim · model-written summary
Wild type and subsite mutants released G6 at 27-40% of the G7 rate.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- G6e0
- Value
- 27-40%e0
- Comparator
- G7 ratee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate”
t0“Barley”
Claim · model-written summary
Wild type and subsite mutants released G8 at 60-70% of the G7 rate.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- G8e0
- Value
- 60-70%e0
- Comparator
- G7 ratee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate”
t0“Barley”
Claim · model-written summary
Wild type and subsite mutants released G9 at 39-48% of the G7 rate.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- G9e0
- Value
- 39-48%e0
- Comparator
- G7 ratee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate”
t0“Barley”
Claim · model-written summary
Wild type and subsite mutants released G10 at 33-44% of the G7 rate.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- G10e0
- Value
- 33-44%e0
- Comparator
- G7 ratee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate”
t0“Barley”
Claim · model-written summary
AMY1-SBD produced G8 at a rate similar to G7.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively”
t0“Barley”
Claim · model-written summary
AMY1-SBD produced G9 at 66% of the G7 rate.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively”
t0“Barley”
Claim · model-written summary
AMY1-SBD produced G10 at 60% of the G7 rate.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively”
t0“Barley”
Claim · model-written summary
G1 through G4 product rates varied substantially among variants.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- shorter productse0
- Direction
- large individual differencese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate”
t0“Barley”
Claim · model-written summary
G5 was consistently a minor product.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- G5e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“G5 was always a minor product”
t0“Barley”
Claim · model-written summary
Long substrate translocation produced G6-G10 largely independently of subsite mutations.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- G6-G10e0
- Qualifier
- longer translocation of substrate in the binding clefte0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations”
t0“Barley”
Claim · model-written summary
Short substrate movements produced individually variable G1-G4 release rates.
- Taxon
- BarleyHordeum vulgaret0
- Outcome
- rates of release of G1-G4e0
- Qualifier
- short-distance movese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“short-distance moves resulting in individually very different rates of release of G1-G4”
t0“Barley”