Studies On The Tissue-Type Plasminogen Activator (t-PA) Fermentation Conditions Produced By Trichoderma Reesei And R-PA Gene Expression In Pichia Methanolica

The dissertation focuses on the fermentation conditions, the fermentation kinetics, the purification and the enzyme characteristics of tissue-type plasminogen activator(t-PA) produced by recombinant Trichoderma reesei.The clone of r-PA cDNA and expression in Pichia methanolica was also investigated.Recombinant Trichoderma reesei strain was screened through dilution-plate method. With t-PA activity as target ,the high-producing Trichoderma reesei (8) with highly genetic stability was selected by shaking flask liquid cultivation of the strains from single colony on plate.The metabolic control mechanism of tissue-type plasminogen activator(t-PA) produced by recombinant Trichoderma reesei was studied.Under the minimal fermentation conditions,the saccharides including sucrose,carboxymethyl cellulose, raffinose,L-sorbose ,lactose ,dissolubility cellulose and maltose could induce the t-PA production. L-sorbose is the best inducer when its concentration is 1.0%. Glucose and its metabolites could produce catabolite repression . Cellobiose could promote the biosynthesis of t-PA when its concentration is low and could produce feedback repression when its concentration is high.The seed medium composition and culture conditions were optimized by single factor experiment and orthogonal experiment. The shaking flask batch fermentation medium composition and conditions were also determined through orthogonal experiment and response surface methodology.The maximum t-PA yield was 3275.26 U/mL,and was improved by thousand times as great as 1.14 U/mL in original fermentation conditions.Based on the optimal conditions obtained in shaking flask, the batch fermentation was performed in 5-liter fermentor. The batch fermentation kinetics of t-PA produced by recombinant Trichoderma reesei were studied based on the experimental data from the fermentation in 5-liter fermentor. The kinetic models of fermentation were established by MATLAB software.Purification process of recombinant t-PA from Trichoderma reesei (8) was explored and established.The recombinant t-PA was purified using ultrafiltration, salting-out, Butyl-Sepharose 4FF hydrophobic interaction chromatography, Sephadex G-25 gel filtration chromatography and Q-Sepharose FF ion exchange chromatography.The purified t-PA was homogeneous examined by SDS-PAGE electrophoresis.The recombinant t-PA was analysed by SDS-PAGE fibrin autography technique .The results showed that there were two kinds of hydrolysis fibrin products in the broth of Trichoderma reesei(8)with 6.6×10~4 and 3.3×10~4 molecular weight respectively.The fibrinolytic characteristic of the recombinant t-PA was also analysed by negative and positive plates,which revealed the t-PA only activated plasminogen to plasmin.there wasnot hydrolysis fibrin product in the broth of host. The preservationtime (4°C) and the freezing-thawing times have much influence on the enzyme activity.The longer preservation time and the more freezing-thawing times ,the more enzyme activity lost. The optimum temperature of the crude and pure recombinant t-PA were 37°C and 45 °C respectively. The optimum pH of the recombinant t-PA was 9.4. The recombinant t-PA was stable in the pH range of 6.4~~8.4 at 37 "C.The isoelectric point of the recombinant t-PA was 3.82 estimated by isoelectrofocusing electrophoresis.The preparation methods of recombinant Trichoderma reesei chromosome DNA were studied.Three kinds of methods of freezing-grinding-CTAB, freezing-grinding-SDS and chloride benzyl-SDS were compared and DNA were detected by agarose gel electrophoresis.The result showed that freezing-grinding -CTAB was a better method.The preparation conditions of the method were optimized.The chromosome DNA prepared by this method meet the requirements of PCR and other molecular biological manipulation.According to the published sequence of r-PA gene, a pair of primers which can amplify the r-PA region was designed. The r-PA gene was amplified by PCR with recombinant Trichoderma reesei chromosome DNA as template, then the r-PA gene was inserted into pMD18-T vector. The recombinant plasmid was used to transform the competent E.coli DH-5a cells. The transformed cells were spread on LB agar plates with ampicillin,X-gal and IPTG to isolation of recombinant strains according to blue-white reaction. A positive clone harboring r-PA gene was identified by restriction enzyme analysis and PCR technique. Collecting recombinant plasmid pMD18-r-PA and sequencing the recombinant plasmid,the sequencing result showed that the cloned 1.1kb fragment of r-PA sequence homology was 99.91% with the r-PA gene published , the amino acid sequence was same as that published.The secreted expression plasmid pMETaA-r-PA of Pichia methanolica was constructed and digested with Pac I and transformed into Pichia methanolica PMAD16 by electroporation.The method of high efficient electrotransformation for pMETotA-r-PA to Pichia methanolica was established.The positive transformants which integrated r-PA gene in their genomes were obtained by screening on MD plates and identified by PCR technique.The Mut phenotypes of these transformants were Muts.Under shake-flask culture and induced using methanol as a carbon source, the extracellular r-PA reached the largest activity of 1675.62U/mL at 72h. The expression products were analyzed by SDS-PAGE.The results indicated that the r-PA from Pichia methanolica PMAD16 was secreted into broth and not glycosylated with 3.96 X 104 molecular weight.