Alpha-amylase blends and methods for using said blends
A technology of amylase and mixture, applied in the field of alpha-amylase mixture and using said mixture, can solve the problems of degeneration, less effective and the like
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0356] Example 1 - Construction of variants
[0357] The variation at position S242 of the mature sequence of AmyS was constructed using a site-directed approach.
[0358] The template used for mutagenesis was methylated pHPLT-AmyS from New England Biolabs (Massachusetts) using dam methylase (see figure 2 ). Degenerate primers (S242F (forward) and S242R (reverse, given below)) were synthesized at Operon (Huntsville, AL) and diluted to 10 μM, both complementary forward and reverse sequences contained 5 'Phosphate group. The sequence of the attached parental α-amylase is SEQ ID NO: 2. Stratagene Quik-Change TM Libraries were created with a multilocus kit (Stratagene, La Jolla CA). The chosen amino acid (ie S242) was randomly replaced with all 19 possible candidates.
[0359] S242 primers for mutagenesis:
[0360] S242F:
[0361] 5'[Phos]GTCAAGCATATTAAGTTTCNNSTTTTTTCCTGATTGGT TG 3'SEQ ID NO: 17
[0362] S242R:
[0363] 5'[Phos]CAACCAATCAGGAAAAAASNNGAACTTAATATGCTTGAC 3'SE...
Embodiment 2
[0372] Example 2 - Expression, purification and characterization of variants
[0373] Colonies were streaked from the microtiter plates of Example 1 and plated on starch plates with 10 ppm neomycin. Plates were incubated overnight at 37°C and single colonies were picked and used to inoculate shake flasks (250 mL shake flasks with 25 mL medium) containing medium (see below) and 20 ppm neomycin. The cultures were incubated at 37° C., 275 rpm for approximately 8 hours (to reach an OD (600 nm) of 2.0). Culture broth was mixed with 50% glycerol in a 2:1 ratio, placed into individually labeled culture vials and frozen at -80°C. Selected alpha-amylases are subsequently produced from these glycerol stores.
[0374] Amylase fermentations were performed in 500 mL shake flasks by incubating minimal MOPS medium (Neidhardt et al., J. Bacteriol. 119(3):736-747, 1974) containing 1% (w / v) soytone for 60 hours at 37°C. The enzyme was purified from the fermentation broth using hydrophobic in...
Embodiment 3
[0376] Example 3 - Determining the nature of the change: heat stress
[0377] This example shows that the variants described herein can have altered properties relative to the parent alpha-amylase. A high-throughput thermostability screen of Geobacillus stearothermophilus alpha-amylase (AmyS) variants was performed.
[0378] Heat stress conditions were studied and selected such that the initial wild-type enzyme showed roughly 40% non-stress activation after heat stress (ie, post heat stress activity / pre heat stress activity was roughly 0.4). Mutant libraries were screened in quadruplicate and potential winners were identified as those mutants that showed residual activity after heat stress that was at least 2 standard deviations higher than the mean residual activity of the original wild-type enzyme.
[0379] Amylase expression was about 100 ppm in the culture supernatant of the expression plate. After 60-65 hours of growth at 37°C in a humidified shaker (250 rpm and 70% rel...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More