Blum_2012_J.Biotechnol_160_214

Reference

Title : Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method - Blum_2012_J.Biotechnol_160_214
Author(s) : Blum JK , Ricketts MD , Bommarius AS
Ref : J Biotechnol , 160 :214 , 2012
Abstract :

alpha-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of alpha-amino beta-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25 degreesC, an observed half-life of 5 min at 30 degreesC, and a "T-50" value, the temperature at which the half-life is 30 min, of 27 degreesC. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a "T-50" value of 34 degreesC (7 degreesC improvement) and 1.3-fold activity compared to wild-type.

PubMedSearch : Blum_2012_J.Biotechnol_160_214
PubMedID: 22426092

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Citations formats

Blum JK, Ricketts MD, Bommarius AS (2012)
Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method
J Biotechnol 160 :214

Blum JK, Ricketts MD, Bommarius AS (2012)
J Biotechnol 160 :214