Background and Objectives: The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production.Materials and Methods: Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al.Results: At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media.Conclusion: Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media.

Treatment of wastewater containing carbohydrates by Pichia saitoi growing on beet molasses was investigated in a well-mixed continuous tank as an alternative to bulking control.The yeast strain that used in this work was isolated from non-alcoholic beverage industrial wastewater, with a view on TOC removal compared with other strains in previous study.In this research the isolated yeast showed high COD and TOC removal at three hydraulic retention times, HRT= 48, 24 and 18 hours.Maximum COD and TOC reductions were obtained at HRT=48 hours, which were 96% and 88%, respectively.The influent COD and TOC were 2500 and 148 mg/l, respectively.The pH maintained for synthetic wastewater was about 9.The changes of pH within the aeration tank at each HRT was monitored.Effects of organic loading rate (OLR) on COD and TOC reduction efficiencies were studied in this work.Aeration rate was between 0.2-0.25 vvm, which was very low in comparison to the other studies.This yeast strain had high settling ability (maximum SVI was observed at 60 ml/g) and also high F/M ratios (maximum of F/M was 2.9 kg COD/kg MLSS.day).

Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems. Materials and Methods: To express LmSTI1 in the methylotrophic yeast Pichia pastoris (P. pastoris), the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0. 5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L. major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively. Results: The expression level was 0. 2% of total soluble proteins. Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization.

Background: Bovine follicle stimulating hormone (bFSH) belongs to the family of glycoprotein hormones that are released from the pituitary gland or the placenta. This hormone is a heterodimer consisting of common a- and specificb-subunit. Bovine FSH is used for reproductive technology such as superovulation in farm animals and polycystic ovary syndrome treatment. Also this hormone is produced for clinical, veterinary purposes and infertility treatment.Recombinant DNA technology provides a useful tool for production of FSH free from hormonal, viral or prions contaminants. The objective of our study was the production of bFSH hormone in Pichia pastoris expression system.Materials and Methods: For this purpose, two ORF strands containing a and b subunits were separately cloned into pTZ57R/T vector and then were subcloned into pPIC9 expression vector and confirmed by PCR and sequencing.The two recombinant plasmids were linearized using the SalI and SacI restriction enzymes in order to generation of His+Mut+GS115. The products were co-transfected into the competent yeast cells by electroporation and examined by PCR.Results: Expression of recombinant bovine FSH in both yeast cells supernatant and pellet was confirmed by SDS-PAGE and Western blotting techniques.Conclusion: Production of recombinant protein in eukaryotic expression systems such as pichia pastoris is a reliable method for producing of therapeutic bovine FSH.Also the detection of the recombinant produced protein by specific antibody confirmed a correct post translated modified structure.

Production of human proteins in Pichia pastoris has significant advantages. However, there is still need for improvement of various stages of its downstream processing like clarification and purification. In fact downstream processes are usually the most critical part of production of biotech products. This work aimed to evaluate the effect of two steps added to the downstream processes of human growth hormone (hGH) production in Pichia pastoris. Firstly, the effect of clarification, with activated carbon, on capture of hGH by ion exchange chromatography (IEC) was investigated. For this purpose, a clarification process using activated carbon was used to remove process contaminants like pigments. The clarified sample was applied to the IEC column and the recovery of hGH, following IEC, was assessed using SDS-PAGE, Bradford protein assay, and area under the curve (AUC). The obtained results showed that the AUC values were 2. 81 and 5. 61 for the with-and without-treatment samples, respectively. Protein recovery of clarified sample with activated carbon was 541 mg in comparison with 328 mg for the sample without treatment. The yield of IEC was also improved from 50. 46% to 83. 23% following treatment with activated carbon. Secondly, the effect of three concentrations of ammonium sulfate in the binding buffer on resolution of hGH upon elution on hydrophobic interaction chromatography (HIC) was investigated. Biological activity was used as the main criterion for evaluation of purified hGH using HIC. The obtained results indicated that by increasing the concentration of ammonium sulfate form 1 to 3 mol/L, resolution of hGH was improved, as the purified fraction using 3 mol/L of ammonium sulfate showed a specific activity of 3. 1IU/mg. So, the results of the present study demonstrated that activated carbon is a promising candidate for efficient clarification of recombinant hGH and for improving the efficiency of the capture step. Therefore, it can be considered by biotech companies as a cost-effective and sustainable clarification procedure of recombinant proteins from high cell density cultures. This study also revealed that 3% ammonium sulfate has a positive effect on the separation of hGH variants with the desired biological activity.