Improvement of the recombinant phytase expression by intermittent feeding of glucose during the induction phase of methylotrophic yeast Pichia pastoris.

PURPOSE: For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. RESULTS: In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02 h before methanol feeding, 1.16% methanol concentration and 40.36µL glucose feeding amount(for 20 mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68 h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. CONCLUSION: Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.

Determination of prostatic fluid citrate concentration using peroxidase-like activity of a peroxotitanium complex.

There have been developed many kinds of methods for detecting citrate in body fluids since citrate is very important physiologically and biochemically. In particular, determination of citrate concentration in prostatic or seminal fluid is useful in early diagnosis of prostate cancer. Recently, a peroxotitanium complex prepared from titanium tetrachloride and hydrogen peroxide has been shown to have peroxidase-like activity which is greatly inhibited by some hydroxyalkanoic acids. Hence, we established a method for determining citrate concentration in prostatic fluid using selective inhibition of citrate on the catalytic activity of the peroxotitanium complex.

Immobilization of Acidithiobacillus ferrooxidans-1333 on the Waste Ore Particles for the Continuous Oxidation of Ferrous Iron.

BACKGROUND: The biooxidation of ferrous iron has a great potential for the regeneration of ferric iron, in operations such as bioleaching, bioremediation. Many natural inorganic materials were investigated for use as supports immobilizing Acidithiobacillus ferrooxidans. The waste chalcopyrite is another natural inorganic material of which particles are easy to prepare from the leached out ore heaps and the source is abundant. OBJECTIVES: The aim of this work is to investigate several characteristics of the particles of waste ore that determines possibility of use as supports for immobilization of Acidithiobacillus ferrooxidans in the packed-bed bioreactor. MATERIALS AND METHODS: Acidithiobacillus ferrooxidans-1333 stored in Korean Centre for Culture Collection was used. The supports were prepared by sieving the particles of 5~30 mm in size out from the waste chalcopyrite ore heap. The cells were immobilized by the successive batch culture method and oxidation rate of the bioreactor was investigated in the continuous flow mode. RESULTS: The cell density of Acidithiobacillus ferrooxidans-1333 immobilized on the particles of waste chalcopyrite was 2.71×108 cells g-1 and the highest oxidation rate of the packed-bed bioreactor was 3.65g.L-1.h-1. Oxidation rate of the bioreactor was less influenced by the concentration of ferrous and ferric iron in the input solution as well as by the aeration rate and dilution rate than other materials mentioned in other previous works. CONCLUSION: The waste chalcopyrite particle is efficient support material for immobilization of Acidithiobacillus ferrooxidans with comparable or superior characteristics to natural inorganic support materials reported before.