Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming

Abstract

Thermostable alkaline pectate lyases can be potentially used for enzymatically degumming ramie in an environmentally sustainable manner and as an alternative to the currently used chemical-based ramie degumming processes. To assess its potential applications, pectate lyase from Bacillus pumilus (ATCC 7061) was cloned and expressed in Escherichia coli. Evolutionary strategies were applied to generate efficient ramie degumming enzymes. Obtained from site-saturation mutagenesis and random mutagenesis, the best performing mutant enzyme M3 exhibited a 3.4-fold higher specific activity on substrate polygalacturonic acid, compared with the wild-type enzyme. Furthermore, the half-life of inactivation at 50 °C for M3 mutant extended to over 13 h. In contrast, the wild-type enzyme was completely inactivated in less than 10 min under the same conditions. An upward shift in the optimal reaction temperature of M3 mutant, to 75 °C, was observed, which was 10 °C higher than that of the wild-type enzyme. Kinetic parameter data revealed that the catalysis efficiency of M3 mutant was higher than that of the wild-type enzyme. Ramie degumming with M3 mutant was also demonstrated to be more efficient than that with the wild-type enzyme. Collectively, our results suggest that the M3 mutant, with remarkable improvements in thermoactivity and thermostability, has potential applications for ramie degumming in the textile industry.

Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming

“…However, the other six mutants exhibiting improved thermostability also had improved specific activity compared to that of the wild-type enzyme (Table 2). Similar results have also been reported for improvement of thermostability in other Pels from Bacillus pumilus (27) and Xanthomonas campestris (42), indicating that the relationship between thermostability and enzyme activity does not have to be opposing.…”

“…Thus, in several cases, thermostability and enzyme activity could not be improved simultaneously (27,46). This phenomenon was also found in this study, and the specific activities of 4 of 10 thermostable mutants obtained from two rounds of random mutagenesis decreased.…”

“…Interestingly, analysis of the intramolecular interactions showed that while some mutants exhibited increased molecular interactions with stabilizing effects, the thermal tolerance of the enzyme was enhanced with the E124I and S271G substitutions, which eliminated several ion pairs or hydrogen bonds; this observation cannot be explained based on our current understanding of the enzyme and enzyme structure (48,50,51). The stabilizing effects of point mutations can be cumulative (27,42,45,47). Indeed, in our study, some further-improved combined mutants were also obtained.…”

“…Although successful approaches for improving the thermostability of pectin hydrolysis enzymes have been reported (27,42,44), improvement of thermostability by protein engineering is still challenging. Directed evolution is a widely used technique for protein engineering in which proteins are improved through iterative cycles of diversity generation and screening; this method has been successfully carried out for various enzymes to improve their thermostability or catalytic activity (27,(45)(46)(47). In this study, BspPelA was successfully engineered by directed-evolution strategies, including random mutagenesis and SSM, in order to improve its thermostability and catalytic efficiency for degumming of ramie fibers.…”

“…In enzymatic degumming, an alkaline environment at a moderate temperature is critical for effective degumming and improvement of fiber quality because the gumlike materials are soluble under these conditions (26); thus, operation of industrial degumming processes is generally carried out at moderate temperatures (40°C to 70°C) and alkaline pH (pH 8 to 11) (27). Therefore, enzymes that are thermostable and active under alkaline conditions are desirable for industrial degumming applications.…”