RESUMO
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.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Boehmeria/química , Gomas Vegetais/química , Polissacarídeo-Liases/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , Primers do DNA , DNA Bacteriano/genética , Estabilidade Enzimática , Escherichia coli/genética , Meia-Vida , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pectinas/química , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
OBJECTIVE: To make the kit with witch to identify Penis et Testis Cervi with molecular taxonomy. METHOD: The mtDNA of sika and red deer from different areas was amplified by PCR and sequenced. Compared with the mtDNA of bovine and horse from witch the false medicines were made, characteristic segments of deer were found. We selected one as the species distinctive PCR primer of deer. RESULT: The kit made up with this primer and related reagents could be used to discern Penis et Testis Cervi from the false medicine. CONCLUSION: It is a scientific, steady, accurate and convenient way to identify Penis et Testis Cervi with molecular taxonomy.