Single amino acid mutation alters thermostability of the alkaline protease from <italic>Bacillus pumilus</italic>: thermodynamics and temperature dependence

Abstract

Dehairing alkaline protease (DHAP) from Bacillus pumilus BA06 has been demonstrated to have high catalytic efficiency and good thermostability, with potential application in leather processing. In order to get insights into its catalytic mechanism, two mutants with single amino acid substitution according to the homology modeling and multiple sequence alignment were characterized in thermodynamics of thermal denaturation and temperature dependence of substrate hydrolysis. The results showed that both mutants of V149I and R249E have a systematic increase in catalytic efficiency (k cat /K m ) in a wide range of temperatures, mainly due to an increase of k 1 (substrate diffusion) and k 2 (acylation) for V149I and of k 2 and k 3 (deacylation) for R249E. In comparison with the wild-type DHAP, the thermostability is increased for V149I and decreased for R249E. Thermodynamic analysis indicated that the free energy (ΔG a°) of activation for thermal denaturation may govern the thermostability. The value of ΔG a°i s increased for V149I and decreased for R249E. Based on these data and the structural modeling, it is suggested that substitution of Val149 with Ile may disturb the local flexibility in the substrate-binding pocket, leading to enhancement of binding affinity for the substrate. In contrast, substitution of Arg249 with Glu leads to interruption of interaction with the C-terminal of enzyme, thus resulting in less thermostability. This study indicates that amino acid residues in the active center or in the substrate-binding pocket may disturb the catalytic process and can be selected as the target for protein engineering in the bacterial alkaline proteases.

Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP)

“…The same observation was demonstrated previously for the variant V149I. 14) M124 and V149 are buried in the structural model, and are close to the substrate binding pocket (Fig. 1(c)).…”

“…The mutant V149I may influence the local flexibility of the substrate-binding pocket. 20) Since the DHAP variant V149I was demonstrated to show an increase of both cold activity and thermostability, 14) combinatorial mutation of V149I with W106K or M124L was constructed to probe the cold activity and thermostability of these mutants. Substitutions of the three sites with Ile, Lys, and Leu, respectively, are presented in the model (Fig.…”

“…23) For the double variants, the catalytic efficiency of W106K/V149I was 3.7-fold higher than that of the wt at 15°C, which was largely due to the higher turnover number (k cat ). Therefore, an accumulative effect of W106K and V149I was integrated into the double-site variant, because the increase in the k cat value of W106K or V149I 14) was enhanced in comparison with that of the wt. The double-site variant W106K/M124L exhibited a decrease in the K m value but an increase in the k cat value when compared with that of the wt, leading to a clear increase in the catalytic efficiency.…”

“…[11][12][13] Based on structural modeling and multiple sequence alignment, the variant V149I demonstrated improved cold activity and an increase in thermostability. 14) In this report, we have integrated V149I with either W106K or M124L, and the resulting double-site variant W106K/V149I was found to show an increase in both specific cold activity and thermostability when compared with that of the wild-type DHAP.…”

“…The desired mutations were confirmed by DNA sequencing and transformed into B. subtilis WB600 competent cells, as described previously. 14) Each of the recombinant B. subtilis WB600 were grown in 1 L Luria-Bertani broth complemented with 1% milk powder to express the protease. After incubation at 37°C for 48 h with vigorous shaking, the supernatant was collected by centrifugation 4600 g for 10 min at 4°C.…”