Enzymes separation from complex mixtures

Basic technology for membrane separation of biomolecules was invented in the United States, but the West Germans and the Japanese lead in its application to separations of enzymes and amino acids from complex mixtures. Japanese... 

Sephadex has been used to a lesser extent in the nucleic acid field. - The G-50 type will separate RNA of high molecular weight from nucleosides or bases. However, Sephadex does not separate the complex mixture of mono- to hexanucleotides obtained by enzymic hydrolysis of RNA. Sephadex-25 has been used as the supporting medium for partition chromatography of yeast-soluble RNA. ... 

In this case, highly specific biochemical interactions provide the means of separation. The stationary phase contains specific groups of molecules which can only adsorb the sample if certain steric and charge-related conditions are satisfied (cf. interaction between antigens and antibodies). Affinity chromatography can be used to isolate proteins (enzymes as well as stmctural proteins), lipids, etc., from complex mixtures without involving any great expenditure. 

When only the enzyme was entrapped in the sol-gel, the rhodium complexes poisoned the enzyme, resulting in low yields, whereas when both enzyme and complex were trapped in separate sol-gel mixtures, good yields were achieved. In one particular case, this procedure resulted in an increase in yield of 1-pentyl undecano-ate from 1 -undecenoic acid and 1 -pentanol by a factor of 6.5, indicating successful site isolation of the mutually interfering catalysts. 

Immobilized metal affinity chromatography has been shown to be effective for isolating proteins from crude mixtures, as well as for selective separations of closely related proteins [2]. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography and the immobilized metalion complexes are much more robust than antibodies or enzymes. These factors make IMAC particularly well suited for scale-up to process scale chromatography. The main scale-up points to be aware of are the degree to which the column is metal saturated, the chelating agent content of the sample, and the potential of leached metal (or its interactions) within the product eluate. 

It has no name, because it is a complex mixture of enzymes. It contains a mixture of one galactosidase and one mannosidase and perhaps more. We have made an attempt in our laboratory to separate the galactosidase activity from the mannosidase activity, but have not been able to do so in a clear-cut fashion. 

---