Novel screening methods--the key to cloning commercially successful biocatalysts.
Providing sufficient biocatalyst to support the demands of multi tonne product supply can be problematical. Here we describe how screening for and cloning a gamma-lactamase overcame biocatalyst supply issues, and greatly improved the actual biocatalytic process. The isolation of an expressing gamma-lactamase clone from a gene library necessitated a combination of classical molecular biology techniques together with innovative screening methods to identify a functional clone. Once isolated the enzyme was characterised with regard to its process performance and proved to be active at 500 g L(-1) substrate. Further development of the recombinant fermentation and downstream processing has resulted in the ability to produce sufficient biocatalyst from one 5001 fermentation to resolve 5 metric tonnes of (+/-)-lactam, whilst simplifying the process chemistry greatly.
Taylor
S J
SJ
Chirotech Technology Ltd., Unit 283, Cambridge, UK. stevetaylor@chirotech.com
Brown
R C
RC
Keene
P A
PA
Taylor
I N
IN
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D016956:Burkholderia cepacia / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular / Q000379:methods*; D020605:Delftia acidovorans / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D015723:Gene Library; D007769:Lactams / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
D007769:Lactams; D011994:Recombinant Proteins; D000581:Amidohydrolases; C401943:gamma-lactamase