| Pectin is a plant polysaccharide that contribute to the structure of plant tissues as a component of the middle lamella and primary cell wall; the backbone of pectin is homopolymer chains of α-1, 4-linked D-galacturonic acid which are partially modified by methyl esterification, the main chain of the polymer also contains rhamnose residues that can be highly substituted by arabinose and galactose side chains. The exsitance of pectin makes it difficult when processing plant, such as reducing the solubility of instant tea, increasing the viscosity of animal feed and hindering the absorb of nutrients by animals, seriously affecting the filtation of pulp in paper making process, influencing the pretreatment of ramie and cotton fabric refining in the textile industry. Pectate lyases(E.C.4.2.2.2) cleave α-1,4-glycosidic linkages by trans-elimination, and break down the pectin. Recently, with the advocation of “green textiles, clean production”, using the bio-refining substitute the traditional alkaline-refining gain more and more attention, as the most effective enzyme in the pre-treatment of cotton fabric, pectate lyases become the reaserch focus.This research selected two pectate lyase genes pel-s(Gene Bank: AB428424) and pel-b(Gene Bank: GU206787) from Gene Bank which belongs to the Bacillus and Paenibacillus. Considering that Pichia pastoris is a mature expression system for the production of heterologous protein, which secretes very little native proteins and produce foreign proteins at high levels, and it has the strong AOX1 promotor, easy of techniques needed for the molecular genetic manipulation and high density fermentation, considering all these advantages, we successfully expressed pectate lyases in P.pastoris. The main contents and results are as follows:(1) This research predict the signal peptide of pel-s and clone it into plasmid p HKA, by using the Saccharomyces cerevisiae α-factor and the putative native signal peptide PF to target it to the secretory pathway, and fine-tuning the AOX1 promoter?s putative transcription factor binding sites, we construct recombinant P.pastoris G/p HKA-α-pel-s, G/p HKA-PF-pel-s and G/p HKA-AOXm-PF-pel-s. Compared to α- factor, the usage of putative native signal peptide PF has improved the activity by 111.66%, then the reformation of AOX1 promotor makes another 51.47% increase, the activity of Pel-s has reached 89.65 U/m L. After optimize the expression elements, we construct the multi-copy strains in order to improve the activity of Pel-s; when induced 144 h, the two-copy strain?s pectate lyase activity reached to 186.96 U/m L, 108.55% higher than the singal-copy strain, and activity of the strain contain four copy pel-s has reached 309.65 U/m L. The result of 50 L fermentation of four-copy stain shows that the activity in the supernatant reached 1729.35 U/m L, 5.58 times of the flask fermentation, and the protein concentration was 9.48 g/L.After purification of Pel-s by affinity chromatography, characterazition of Pel-s has been detected, the optimal temperature and p H is 80 oC and 10.0, after treated with different buffer range from p H 3-11 for 16 h, the residual activity is still retain more than 90%; and when incubated Pel-s at 30-50 °C for 240 min, the activity is slightly declined, when incubated at 60 oC for 180 min, about 40.71% activity is maintained, while when treated at 70 oC for 180 min, only 16.81% is remained. The effects of the metal ions show that the two Pectate lyases rely on Ca2+ when break down pectin.(2) Use the same methods above to construct the secreted recombinant P.pastoris G/p HKA-SP-pel-b, p HKA-α-pel-b and G/p HKA-AOXm-α-pel-b. Enzyme production curve tells that, when induced 168 h by methanol, the use of α- factor makes the enzyme activity up to 307.25 U/m L, 14.65 times of the SP, the reform of AOX1 improve the activity another 40.72%, up to 432.36 U/m L, higher than reported at present.The optimal temperature and p H of recombinant Pel-b is 60 oC and 10.0, respectively. The activity was slightly affected when Pel-b was treated with different buffer range from p H 9-12 for 16 h. And the residual activity was still higher than 80% when incubated Pel-b at 30-50 °C for 300 min, even incubate at 60 oC for 240 min, there is still 40.75% activity maintained, proved that Pel-b has good thermal stability, can meet the requirement for temperature and alkali resistance when used in refining cotton fabrics. |
PDF Full Text Request