Enzymes for starch processing
A technology of amylase and amino acid, applied in the fields of enzymes, applications, genetic engineering, etc., can solve problems such as energy consumption
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Examples
Embodiment 1
[0186] Example 1: Cloning of Carbohydrate Binding Module (CBM) from Aspergillus kawachii
[0187] In order to clone the carbohydrate-binding module (CBM) with linker from Aspergillus kawachii, primer CBM1 (SEQ ID NO: 1 ) and CBM2 (SEQ ID NO: 2). CBM1 and CBM2 contain BamHI and SalI sites, respectively.
[0188] CBM1: 5'-gaagggatccgatttttactagtacatccaaagccaccac-3'
[0189] CBM2: 5'-tttgtcgacctacctccacgtatcaaccaccgtctcc-3'
[0190] Using primers CBM1 and CBM2, a PCR reaction was performed using genomic DNA from Aspergillus kawachii (IFO4308) as a template. Reaction components (1 ng / microL of genomic DNA, 250 mM each of dNTPs, 250 nM each of primers, 0.1 U / microL of Taq polymerase (Roche Diagnostics, Japan) in 1X buffer) were mixed and PCR was performed under the following conditions.
[0191] steps
temperature
time
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94℃
92℃
55℃
72℃
72℃
4℃
2 minutes
1 minute
1 minute
1 mi...
Embodiment 2
[0194] Embodiment 2: express described hybrid enzyme in Aspergillus niger
[0195] The cDNA sequence producing the A. niger acid stable alpha-amylase gene and the cDNA cloning of the A. niger acid stable alpha-amylase are described in WO 8901969 (Examples 1 and 3). A PCR reaction using the A. niger acid-stabilized alpha-amylase cDNA clone as a template was performed using primers (SEQ ID NO: 3) and (SEQ ID NO: 4) to introduce a BamHI site and a SpeI site, respectively.
[0196] (SEQ ID NO: 3): 5'-tttggatccaccatgagattatcgacttcgagtctcttc-3'
[0197] (SEQ ID NO: 4): 5'-tttactagtagcagcagcagttgtggtcgtggttgttc-3'
[0198] Reaction components (1 ng / microL of template DNA, 250 mM of each dNTP, 250 nM of each primer, 0.1 U / microL of Taq polymerase (Roche Diagnostics, Japan) in 1× buffer) were mixed and PCR was performed under the following conditions .
[0199] steps
temperature
time
1
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3
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5
6
94℃
92℃
55℃
72℃...
Embodiment 3
[0205] Example 3: Performance of hybrid enzymes in SSF of non-gelatinizing starch
[0206] The relative performance of A. niger acid-stabilized alpha-amylases and A. niger acid-stabilized alpha-amylases with additional sugar-binding modules was evaluated by small-scale SSF (simultaneous saccharification and fermentation) fermentation. The applied doses were 0.3, 0.5 and 1.0 AFAU / g DS alpha-amylase supplemented with purified A. niger glucoamylase at a dose of 0.5 AGU / g DS. Briefly, approximately 1.9 g of ground corn (yellow #2 dent corn ground in a pilot scale hammer mill and passed through a 2mm screen, 11.2% moisture content) was added 16ml polystyrene tubes (Falcon 352025). Add an appropriate amount of 0.02N H containing 3 mg / ml penicillin 2 SO 4 The solution was brought to the dry solids level (DS) of 34% and the pH to 5.0. Treatments were repeated six times.
[0207] After adding the enzyme, the tube was treated with 4.74×10 8 Yeast cells / ml inoculum. Tubes were fit...
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