Directed Evolution of E. coli Alkaline Phosphatase Towards Higher Catalytic Activity

Abstract

Directed evolution was used to enhance the catalytic activity of E. coli alkaline phosphatase (EAP). Through two rounds of error-prone PCR and one round of DNA shuffling followed by a rapid, sensitive screening procedure, several improved variants were obtained. Their enzymatic kinetic properties, thermal stabilities and possible mechanism for the improvement were investigated. In 1.0 M Tris buffer, the specific activity of the most active EAP variant S2163 was 1500 units/mg protein, showing it to be 3.6 times more active than the D101S parent enzyme and Â/40 times more active than the wild-type EAP. At the same time, the K m value of the S2163 variant decreased to 1491 mM from the 2384 mM of the D101S. As a result, the k cat /K m ratio of this variant showed a 5.8-fold enhancement over that of D101S parent enzyme. Three activating amino acid substitutions, K167R, G180S and S374C, which were located far away from the center of the catalytic pocket, were identified by sequencing the genes encoding evolved enzymes. Possible explanations for the improvement of activity were analyzed.

Characterization of a monomeric heat-labile classical alkaline phosphatase from Anabaena sp. PCC7120

“…This might be due to lower K m value. A similar observation was previously made with EAP in our lab: substitution of Lys167, which is adjacent to Arg166, resulted in a more positively charged environment around the substrate binding pock et, which could stabilize the transition state complex and accelerate the rate of hydrolysis of the covalent E-P intermediate [47]. These results clearly show that the active sites of PhoA are highly conserved in AP A and fur ther support the conclusion that AP A is a classical PhoA type alkaline phosphatase, though difference do exist between this enzyme and those from other species.…”

Introduction to in vitro culture and callus initiation in Salvia hispanica L. (chia)

“…Для проведення ренатурації rhIL7-BАРmut застосовано метод плавного розведення. Додаткові речовини, які забезпечують інгібування агрегації, а також співвідношення окисно-відновних реагентів для коректного формування дисульфідних зв'язків підбирали відповідно до даних літератури (Yamaguchi et al, 2014;Xu et al, 2003). Додавання аргініну у концентрації 0,1-1 М дозволяє знизити агрегацію білка та підвищити його розчинність.…”

Directed evolution of the 5'‐untranslated region of the phoA gene in Escherichia coli simultaneously yields a stronger promoter and a stronger Shine‐Dalgarno sequence

“…The screening of mutant libraries was performed as described before [14]. The clone with the highest cellular EAP activity was isolated and subjected to sequence analysis.…”

“…been successfully used in our previous study [14] and was again adopted in this study. Both screening and identification of mutations with the desired property could be accomplished by spectrometric assays.…”

“…The enzyme has been well characterized and widely applied [11][12][13]. Our previous study of directed evolution in the coding sequence has generated one mutant enzyme with nearly 40-fold increased activity [14], which has been successfully applied as a sensitive marker for biochips [15][16][17][18]. In this study, directed evolution was utilized to improve the regulatory sequence and the coding sequence of EAP.…”