Expression Of Pectinases Of Aspergillus In Escherichia Coli

Pectinases are mainly used in the food industry to clarify fruit juices and wine, improve oil extraction, remove the peel from the citrus fruit, increase the firmness of some fruits and degum fibres. Aspergillus fungi, as the familiar fungi could produce pectinases, used for the production of traditional fermented foods, only could produce less pectinases under natural conditions. So far only a few of pectinases expressed in yeast or E.coli were reported but they did not show higher activity. Pectate lyase and polygalacturonase have an extensive source, reported in the plant, fungi and bacteria. Pectate lyase A (PelA) of Aspergillus nidulans and Polygalacturonase A (PGA) of Aspergillus oryzae were successfully expressed in Escherichia coli.The cDNA of mature PelA (without signal peptide) was synthesized with specific primers from total RNA of Aspergillus nidulans by RT-PCR. PelA cDNA was ligated into pET-28a(+) expression vector, creating plasmid pET-28a(+)-pelA. The plasmid was transformed into E. coil Turner (DE3) plac I cells to express PelA heterogeneously. For improving the efficiency of PelA expression in E. coil, the conditions for expression of the PelA in E. coli were optimized. E. coil Turner (DE3) plac I cells with pET-28a(+)-pelA were first cultivated at 37℃, 220 rpm until OD_600nm reached about 0.8. Then, cultivation broth was added with 0.5 mM IPTG and incubated at 20^C, 170 rpm for other 60 h for induced-expression of PelA.The recombinant PelA was effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 UmL^-1 medium, which is 3600-fold of the activity of PelA produced in culture of A. nidulans and also superior than known recombinant expression amount of PelA reported by other researchers. Low-esterified pectin (pectin esterified approx 30% or ploygalacturonic acid) was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5-10 at 50℃. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺, and Fe³⁺ ions stimulated the pectate lyase activity, but Cu2+ and Zn2+ inhibited it. The recombinant PelA had a V_max of 77μmol min^-1mg^-1 and an apparent K_m of 0.50 mg m1^-1 for polygalacturonic acid. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.The cDNA of mature PGA (without signal peptide) were synthesized with specific primers from total RNA of Aspergillus oryzae by RT-PCR. PGA cDNA was ligated into pET-28a(+) .expression vector, creating plasmid pET-28a(+)-pgA. The plasmid was transformed into E. coil Turner (DE3) placâ… cells to express PGA heterogeneously. For improving the efficiency of PGA expression in E. coli, the conditions for expression of the PGA in E. coil were optimized. E. coli Turner (DE3) placâ… cells with pET-28a(+)-pgA were first cultivated at 37℃, 220 rpm until OD_600nm reached about 0.8. Then, cultivation broth was added with 0.5 mM IPTG and incubated at15^C, 170 rpm for other 24 h for induced-expression of PGA. Our data showed that the activity of recombinant expressed PGA could reach to 70 UmL^-1 medium, which is 87.5-fold of the activity of PGA produced in culture of A. oryzae and superior than known recombinant expression amount of PGA reported by other researchers. As same as PelA, Iow-esterified pectin (pectin esterified approx 30% or ploygalacturonic acid) was the optimum substrate for the PGA, whereas higher-esterified pectin was hardly cleaved by it. PGA exhibited its optimum level of activity over the range of pH 5.0-5.5 at 50℃. Cu²⁺ and Zn²⁺ inhibited the PGA activity. PGA also efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.