High-level Production Of Glucose Oxidase By Recombinant Pichia Pastoris And High-thermostability By Computer-simulation

Glucose oxidase (GOD), oxidizing glucose to glucuronic acid, exists widely in animals?plants and microorganisms. It is widely used in food?pharmaceuticals and industry. In this work, GOD was successfully expressed in Pichia pastoris. To further improve the expression of GOD through the integration of molecular chaperones and optimizing fermental conditions in 5 L bioreactor. Finally improving the thermostability of GOD by computer simulation method.The main conclusions are as follows:In this work, the strain, X33/pPIC9k-GOD, was successfully constructed in Pichia pastoris. The protein disulfide isomerase (PDI) and Vitreoscialla hemoglobin (VHb) were co-expressed with GOD separately and simultaneously. Compared with wild type, the strains that were expressed with PDI, VHb and PDI-VHb were able to increase GOD activity with 1.2,0.4 and 1.5 fold and their activity reached 29 U/mL,18 U/mL and 33 U/mL respectively. The strain, X33/pPIC9k-GOD, was fermented in 5 liter bioreactor, and its activity reached 302 U/mL with a yield of 2700 mg/L total soluble protein concentration. The strain, which was co-expressed PDI-VHb, was fermented in 5 liter bioreactor, and its activity reached 603 U/mL with a yield of 6100 mg/L total soluble protein concentration.In base of the strain, X33/pPIC9k-GOD, the optimization of fermental process in 5 L bioreactor was studied. The results showed that optimized fermentation conditions of GOD were pH 6.0,30?, adding 1 mM VB2 at 90 h after inducing and induced by 20:1 (W/W, methanol:sorbitol) co-feeding of methanol/sorbitol. The activity of the strain, X33/pPIC9k-GOD, using optimized fermentation conditions, reached 456 U/mL with a yield of 5100 mg/L total soluble protein concentration. While the activity of GOD, expressed by the strain X33/pPIC9k-GOD/pPICZ-PDI-VHb, reached 716 U/mL with a yield of 7100 mg/L total soluble protein concentration.Using computer-simulation design methods, the thermostability of GOD was studied. Three amino acid mutations S16K, Q243H and H477Y developed the thermostability of GOD with 2?,1.5? and 2? respectively and their thermostability reached 60.5?,60 ??60.5?. The activity of GOD was not affected by S16K and H477Y while Q243H mutation increased the activity of GOD with 0.2 fold, and its GOD activity reached 15 U/mL. The specific activity of GOD was not influenced by three mutations. The strain, X33/pPIC9k-GOD/mut3, containing three mutations, was fermented in 5 L bioreactor with the optimized conditions, and its acticvity reached 504 U/mL with a yield of 5800 mg/L total soluble protein concentration. Compared with wild type, the GOD thermostability was developed 4.5? and reached 63?,.