Enhanced thermostability of keratinase by computational design and empirical mutation

Abstract

Keratinases are proteolytic enzymes capable of degrading insoluble keratins. The importance of these enzymes is being increasingly recognized in fields as diverse as animal feed production, textile processing, detergent formulation, leather manufacture, and medicine. To enhance the thermostability of Bacillus licheniformis BBE11-1 keratinase, the PoPMuSiC algorithm was applied to predict the folding free energy change (ΔΔG) of amino acid substitutions. Use of the algorithm in combination with molecular modification of homologous subtilisin allowed the introduction of four amino acid substitutions (N122Y, N217S, A193P, N160C) into the enzyme by site-directed mutagenesis, and the mutant genes were expressed in Bacillus subtilis WB600. The quadruple mutant displayed synergistic or additive effects with an 8.6-fold increase in the t 1/2 value at 60 °C. The N122Y substitution also led to an approximately 5.6-fold increase in catalytic efficiency compared to that of the wild-type keratinase. These results provide further insight into the thermostability of keratinase and suggest further potential industrial applications.

Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176

“…The loop area of the BgAP surface was replaced by substitutions to negatively charged amino acids, which contributed to the increase of the thermostability, probably through increasing ionic and hydrogen bond interactions in mutated BgAP. The half-life of a keratinase at 60°C from B. licheniformis BBE11-1 was increased 8.6-fold by four amino acid substitutions (N122Y, N217S, A193P, N160C) [23]. Increase in the thermostability was explained to be the result of increased interactions, such as hydrophobic interaction, cation-pi interaction, and hydrogen bond, in the mutant.…”

“…The 122 th residue of mutant (N122Y) was located 6.42 Å, 6.63 Å, and 2.45 Å from Asp 32 , His 63 , and Ser 220 , respectively. Some intramolecular interactions among the aromatic 122 th residue, the active site (Asp 32 , His 63 , Ser 220 ), and substrate influenced to increase the catalytic efficiency [23], whereas other substitutions (N217S, A193P, and N160C), which were located far from the active site, did not significantly change the K m or k cat [23]. For a serine alkaline protease from B. pumilus CBS, the catalytic efficiency was increased 42-fold by mutations (L31I/T33S/N99Y) [10].…”

Microbial Enzymes with Special Characteristics for Biotechnological Applications

“…[23] have reported the enhanced thermostability of a preparation of keratinase by computational design and empirical mutation. The quadruple mutant of Bacillus subtilis has been characterised to exhibit the synergistic and additive effects at 60 °C with an increase of 8.6-fold in the t1/2 value.…”

Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

“…PoPMuSiC evaluates the stability changes resulting from all possible mutations and returns a report containing a list of the most stabilizing mutations or destabilizing mutations, or the mutations that do not affect stability [18]. This algorithm has been proved useful in the design of stabilized point mutations of tobacco etch virus protease [19], pyruvate formate lyase [20], feruloyl esterases [21], glycerol dehydratase [22], keratinase [23], alkaline ␣-amylase [24] and some other enzymes.…”