Hybrid enzymes
A hybrid and amylase technology, applied in the field of hybrid enzymes, can solve problems such as stickiness and loss of elasticity of baked products
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Embodiment 1
[0191] Example 1: Construction of a hybrid between an endoamylase and a CBM from AMY1048
[0192] Amylase AMY1048 is a wild-type Bacillus amylase composed of a 484 amino acid catalytic segment and an additional 101 aa CBM20 segment. The DNA sequence encoding AMY1048 is included in SEQ ID NO:1 and includes the mature AMY1048 sequence as SEQ ID NO:2. In SEQ ID NO:1, the CBM is defined as amino acid residues 485 to 586, which correspond to nucleotides 1540-1845 in SEQ ID NO:2. The CBM-containing amylase can be expressed from a construct similar to that described for other amylases, i.e., inserted into a vector under the control of a constitutively active promoter and flanked by the signal for the Bacillus licheniformis endoamylase sequence (SEQ ID NO: 15) and terminator sequence.
[0193] Replacement of the catalytic fragment of the AMY1048 endoamylase with the catalytic domain of another endoamylase, resulting in a hybrid of the CBM from AMY1048 with a novel endoamylase amplif...
Embodiment 2
[0221] Construction of hybrid amylases with sugar-binding domains
[0222] The catalytic segment of the Bacillus flavus endoamylase AMY1048 can be further divided into a central AB-domain containing the catalytic center and a C-domain located C-terminal to the catalytic domain but preceding the CBM. In SEQ ID NO:2, the catalytic core domain consists of the first 397 amino acid residues, the C domain is defined as amino acid residues 398 to 484 and the CBM is defined as amino acid residues 485 to 586. In SEQ ID NO: 1, the signal sequence is encoded by nucleotides 1 to 87, the catalytic core domain is encoded by nucleotides 88-1278, the C domain is encoded by nucleotides 1279-1539, and the CBM is encoded by nucleotides 1540 -1845 encoding.
[0223] The CBM-containing amylase can be expressed from a vector construct similar to that described in Example 4 of WO0060060A2, i.e. the amylase gene is inserted into a vector under the control of the amylase promoter, flanked by a signal...
Embodiment 3
[0235] Example 3: Determination of exo-endo activity improvement factor (EIF)
[0236] EIF is a measure of the increased ratio of exo / endo activity relative to the parent enzyme, ie EIF=(exo / endo of variant) / (exo / endo of parent enzyme). If EIF > 1 the enzyme has an increased exo / endo activity ratio compared to its parent enzyme. EIF can be based on one of the following methods.
[0237] EIF1 endo-activity assay : The Phadebas Amylase Test (Pharmacia Diagnostics) was performed according to the manufacturer's recommendations and the units of endo-activity were calculated from the formula provided, where the natural logarithm of this activity is equal to N, where N = square root of A + [B + C * ln(Abs)] . Abs is the absorbance at 620 nm, A=-13.3235, B=243.3293, and C=26.73797.
[0238] Exo-activity assay : 50 μl of 50 mM sodium citrate, 5 mM CaCl 2, pH 6.5 was mixed with 25 microliters of enzyme in the same buffer and 25 microliters of Betamyl substrate was dissolved acco...
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