1. The development and application of a new affinity partitioning system for enzyme isolation and purification
A L Nguyen, J H Luong Enzyme Microb Technol. 1990 Sep;12(9):663-8. doi: 10.1016/0141-0229(90)90005-b.
A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.
2. Rapid and efficient one-step purification of a serralysin family protease by using a p-aminobenzamidine-modified affinity medium
Shangyong Li, Linna Wang, Shengxiang Lin, Juan Yang, Zibin Ma, Yuejun Wang, Junzhong Liu, Jianhua Hao, Mi Sun J Sep Sci. 2017 May;40(9):1960-1965. doi: 10.1002/jssc.201601375. Epub 2017 Apr 18.
The metalloproteinase MP belongs to the serralysin family, which is involved in important functions such as nutrient acquisition and infection pathogenesis. Serralysin proteases in highly purified form are commonly used at the industrial level with several purposes. In this study, we set up an efficient and rapid purification protocol for MP using a p-aminobenzamidine-modified affinity chromatography. The affinity medium was synthesized by using p-aminobenzamidine as affinity ligand immobilized via cyanuric chloride spacer to Sepharose 6B sorbent carrier. According to the adsorption analysis, the dissociation constant Kd and theoretical maximum adsorption Qmax of this medium were 24.2 μg/mL and 24.1 mg/g wet sorbent, respectively. The purity of MP was assessed by a high-performance liquid chromatography on a TSK3000SW column and sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealing values of 98.7 and ~98%, respectively. The specific activity of purified MP was 95.6 U/mg, which is similar to values obtained through traditional purification protocols. In conclusion, our protocol could be easily employed for the rapid isolation of MP with high purity, and could be implemented for other serralysin family proteases.
3. Three-component reaction of tautomeric amidines with 3-ferrocenylmethylidene-2,4-pentanedione. Formation of polymeric coordination complexes of potassium ferrocenyl-(hexahydro)pyrimidoxides
Elena I Klimova, Marcos Flores-Alamo, Tatiana Klimova, Sandra Cortez Maya, Irina P Beletskaya Molecules. 2013 Dec 20;19(1):41-54. doi: 10.3390/molecules19010041.
Acetamidine hydrochloride and p-aminobenzamidine dihydrochloride interact with 3-ferrocenylmethylidene-2,4-pentanedione at 80-82 °C in the presence of K2CO3 in the water-alcohol medium in two tautomeric forms (the amidoimine and enediamine ones) with formation of mixtures of pyrimidine and piperidone derivatives and polymeric coordination complexes of potassium ferrocenyl(hexahydro)pyrimidoxides. The structure of the resultant compounds is elucidated on the basis of IR, 1H- and 13C-NMR spectroscopy, mass spectrometry and elemental analysis data. The crystal structures of 6-ferrocenyl-4-hydroxy-4-methyl-2-piperidone, potassium 6-ferrocenyl-4-methyl-2-methylidene(hexahydro)pyrimidin-4-oxide and 2-(4-aminophenyl)-4-ferrocenyl-6-methyl-pyrimidine were determined by X-ray analysis of suitable single crystals.
