title: 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.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China.
authors.forename: Chaoning
authors.initials: C
authors.lastname: Liang
authors.forename: Xiwu
authors.initials: X
authors.lastname: Gui
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhou
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Xue
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
authors.forename: Shuang-Yan
authors.initials: SY
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D032402:Boehmeria / Q000737:chemistry*; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010368:Pectins / Q000737:chemistry; D053149:Plant Gums / Q000737:chemistry*; D011133:Polysaccharide-Lyases / Q000737:chemistry*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D053149:Plant Gums; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; C003181:polygalacturonic acid