Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25.
Directed evolution coupled with a high-throughput robotic screen was employed to broaden the industrial use of the maltogenic alpha-amylase Novamyl from Bacillus sp. TS-25. Wild-type Novamyl is currently used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. However, the enzyme is rapidly inactivated during the baking process of bread made with low pH recipes and Novamyl thus has very limited beneficial effect for this particular application. In an effort to improve the performance of Novamyl for low pH bread applications such as sourdough and rye, two error-prone PCR libraries were generated, expressed in Bacillus subtilis and screened for variants with improved thermal stability and activity under low pH conditions. Variants exhibiting improved performance were iteratively recombined using DNA shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10 degrees C increase in thermal stability at pH 4.5, one of which demonstrated substantial anti-staling properties in low pH breads.
Jones
Aubrey
A
Novozymes Inc., 1445 Drew Avenue, Davis, CA 95616, USA. aujo@novozymes.com
Lamsa
Michael
M
Frandsen
Torben P
TP
Spendler
Tina
T
Harris
Paul
P
Sloma
Alan
A
Xu
Feng
F
Nielsen
Jack Bech
JB
Cherry
Joel R
JR
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001939:Bread / Q000382:microbiology; D032621:DNA Shuffling; D019020:Directed Molecular Evolution / Q000379:methods; D004795:Enzyme Stability; D005516:Food Microbiology; D015723:Gene Library; D024861:Genetic Enhancement / Q000379:methods; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D006361:Heating; D006863:Hydrogen-Ion Concentration; D015202:Protein Engineering; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
D006026:Glycoside Hydrolases; D000516:alpha-Amylases; C120909:glucan 1,4-alpha-maltohydrolase