Optimal Extraction,Purification and Activity Determination of Phytase from Triticale

[ Objective] This study aimed to optimize the extraction conditions of high-activity phytase from triticale. [ Method] Food and forage triticale 209 was used as an experimental material to investigate the optimal extraction conditions, including pH, solid-liquid ratio, extraction duration and active agent concentra- tion. The extracted phytase was purified with salting-out concentration method for SDS-PAGE eIectrophoresis Total protein content was measured using Bradford method; phytase activity was measured using vanadium ammonium molybdate method in accordance with the national standard GB/T 18634 -2009. [ Result] Phytase activity reached the highest under extraction conditions of pH 5.0, solid-liquid ratio 10, room temperature, shaking speed 200 r/min and shaking duration 1 h, without addition of active agems. [ Condusion] This study improved the extraction technology of phytase from wheat plants and was suitable for practical ap- plication.

Methodological approach to the isolation of functionally active proteins from the tissues of marine hydrobionts: an example of Adamussium colbecki

Enzymes from cold-adapted organisms have significant application potential. Because of their unique properties they have been found to be useful in various industries. Despite indisputable practical interest, cold active enzymes also represent a valuable model for fundamental research into protein folding and catalysis. Many investigators have focused their attention on marine hydrobionts, which are growing in importance as a promising source of enzymes. The nature of the source not only determines the availability and the cost of biomolecules of interest but also determines the choice of method for their extraction. A simple and convenient methodological approach of two-stage extraction of proteins has been tested on the Antarctic marine hydrobiont--Adamussium colbecki. This method extracts enough effective protein directly from primary raw materials, as well as when using leftover crude precipitates. The electrophoretic pattern of proteins showed the presence of molecules in a wide range of molecular weights in the samples of A. colbecki after the first and the second stage of extraction. The general proteolytic activity in the first and the second extracts were examined using a zymogram technique. Our experiments revealed that the second extract of A. colbecki contained thermo stable protease exhibiting a molecular weight of 95 kDa in a gelatin zymogram. Further biochemical assays, using different substrates, were conducted to partially identify the types of hydrolases present in the first and the second extracts. Our results revealed the presence of enzymes with collagenolytic and some amylolytic activities preserved in the second extracts. But no esterase or amidase trypsin-like activities were found in the second extract, in contrast to the first extract where this type of activity was significant.

Expression of Drosophila melanogaster acetylcholinesterase(DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides

Acetylcholinesterase(AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method.An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris.The pPIC9 K-Drosophila melanogaster acetylcholinesterase(DmAChE)-like expression vector was constructed by codon optimization of this mutant strain,which was transformed into P.pastoris GS115,and positive clones were selected on yeast peptone dextrose(YPD) plate with G418 at 4.0 mg/mL.The GS115-pPIC9 K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h,with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) pattern of the fermentation supernatant.After preliminary purification by ammonium sulfate precipitation,the enzyme activity was detected to be 76.9 U/(mL·min).In addition,the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.