| Thermostable alkaline pectate lyases have application used in the field of medicine, textile, plant fiber processing, oil extraction, and treatment of industrial was tewater containing pectinacious material. In the technology of enzymatic extraction, pectinase can destroy the pectin in the cell wall of plant, contribute to release the effective components of traditional Chinese medicine.Refining Chinese medicine effective component with pectate lyases is not only economical and environment-friendly, but also more high-efficiency. Alkaline pectate lyase from alkaliphilic Bacillus pumilus has high activity and high optimal reaction pH, but it is not stable in high temperature conditions.Fructus Lycii is a valuable and traditional Chinese food-medicine with immune regulation, anti-aging, anti-tumor, improving eyesight and other physiological functions. In recent years it was found that the medical and health care function of it was related to its polysaccharide content. So many scholars extracted polysaccharide from Fructus Lycii with different methods, such as water extraction, ultrasonic extraction, alkaline extraction and so on.Thermostable alkaline pectate lyases can be potentially used in the field of medicine. Refining Chinese medicine effective component with pectate lyases is not only economical and environment-friendly, but also more high-efficiency.In this paper, improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus by directed evolution for extraction of lycium barbarum polysaccharides. Application value of pectate lyases is improved. It provides a new method for extracting active ingredients of Chinese medicine. The results were as follows:1) Alkaline pectate lyase gene cloning Pectate lyase from Bacillus pumilus (ATCC 7061) was cloned to construct the recombinant plasmid pET28a-pel, the recombinant molecular weight is 37 kDa.2) Constructing DNA library and screening mutants Six site-directed mutants were obtained by site directed mutagenesis and Megawhop PCR. The appropriate mutational site R227 was found. For site-saturation mutagenesis on R227, we get a better mutant R227W than wild-type enzyme. A new random mutagenesis library was generated by error-prone PCR and Megawhop using the recombinant plasmid R227W as template. Two better mutants 12E4 (S145C, R227W),13E8 (T206A, R227W) were obtained.12E4 was better than R227W in thermo activity.13E8 was more stable than R227W in high-temperature environments. M3 mutant(S145C, T206A, R227W)was obtained by combining the three mutation sites by site-directed mutagenesis method.3) The contrast of wild-type alkaline pectate lyase and the mutant 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℃ 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℃, was observed, which was 10℃ higher than that of the wild-type enzyme. Kinetic parameter kcat/Kmdata revealed that the catalysis efficiency of M3 mutant was higher than that of the wild-type enzyme.4) Enzymatic extraction of lycium barbarum polysaccharides Wild-type pectate lyase and mutant M3 was applied to extract lycium barbarum polysaccharides. From the efficiency of extraction of lycium barbarum polysaccharides, M3 is better than the wild-type pectate lyase. At 60 degrees, pH10.0 for 2 hours, the extraction rate of M3 was 4.3%, wild-type pectate lyase was 3.0%,buffer was 1.8%,water was 1.0%. Adding alkaline pectinase in alkaline environment is good for extraction of lycium barbarum polysaccharides... |
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