Method and apparatus for continuous flow isoelectric focus for purifying biological substances

Abstract

The invention provides a continuous flow apparatus and method for purifying charged substances from solutions or suspensions by isoelectric focusing on an industrial scale. The apparatus comprises a separating chamber, a pair of vertical electrodes ( 9 ) located at the extremes of the apparatus, an anion- or a cation-selective membrane ( 10 ) located near each of the electrodes ( 9 ) for separating electrode spaces from a central part of the apparatus, one or more outflow ports ( 12 ) for separating liquid fractions at the upper part of the chamber, two secondary outflow ports ( 11 ) being provided in the upper part of each of the electrode spaces, one or more vertical wall(s) ( 6 ) between each of said outflow ports partitioning the chamber at least at the height of said outflow ports ( 11,12 ), and a plurality of vertical permeable partition walls ( 13 ) to enable a convection-free upward flow. The apparatus invention does not require additional amphoteric buffering solutions to establish a suitable pH gradient, and can be used for purification of polynucleotides, amino acids, peptides, proteins, organic solvents and beverages.

What is claimed is:  
     
         1 . A purified hydrolytic enzyme or enzyme mixture derived from earthworms, especially from the species  E. foetida,  corresponding to eisenase, fellulase, fetilase, fetipase, and wormase, having an isolectric point determined by isoelectrofocusing of 8.3, 7.0, 6.5, 3.2, and 2.8, respectively, and having a molecular weight determined by SDS-PAGE of 17, 67, 36, 18 and 31 kDA, respectively, or a mixture of two or more thereof.  
     
     
         2 . A method for preparing a thrombolytic drug or medicine comprising formulating an enzyme according to  claim 1  in a pharmaceutical composition.  
     
     
         3 . A method for enhancing the sugar content during the malting process of beer comprising applying an enzyme according to  claim 1  to a beer malting process.  
     
     
         4 . The purified hydrolytic enzyme mixture according to  claim 1 , which is a mixture of eisenase, fellulase, fetilase, and wormase.  
     
     
         5 . A method for remediation of soil and subsoil water, oil spills in seawater, for wastewater treatment, and for cleaning of sea ships and tankers, comprising applying a composition comprising a mixture of the enzymes wormase, eisenase, fetipase and fetilase according to  claim 4  at a ratio wherein each of the enzymes wormase, cisenase, fetipase and fetilase is in a range of 15-40, with the mixture being 100 parts total, to a substance to be remediated.  
     
     
         6 . A method for cleaning slaughterhouses, milking machines, car washs, washing machines, bathrooms, floors, pipes or equipments, comprising applying a composition comprising a mixture of the enzymes wormase, eisenase, fetipase and fetilase according to  claim 4  at a ratio wherein each of the enzymes wormase, eisenase, fetipase and fetilase is in a range of 20-30 parts, with the mixture being 100 parts total, to a surface to be cleaned.  
     
     
         7 . A method for odor treatment or reducing ammonia emission, comprising applying a composition comprising a mixture of the enzymes wormase, cisenase, fetipase and fetilase according to  claim 4  in a ratio wherein each of the enzymes wormase, eisenase, fetipase and fetilase is in a range of 10-55 parts, with the mixture being 100 parts total, to organic waste and/or animal litter.  
     
     
         8 . A method for preparing a prebiotic or (a prebiotic or a growth promoter) for supplementing animal feed, comprising formulating a mixture of the enzymes wormase, eisenase, fetipase and fetilase according to  claim 4  in a ratio wherein each of the enzymes wormase, cisenase, fetipase and fetilase is in a range of 10-40 parts with the mixture being 100 parts total, in a prebiotic or supplement to animal feed.  
     
     
         9 . A method for preparing a shampoo or toothpaste, comprising formulating a mixture of the enzymes wormase, eisenase, fetipase and fetilase according to  claim 4  in a shampoo or toothpaste, wherein the enzymes are present in equal parts.