Immobilized interfacial enzymes of improved and stabilized activity

Abstract

Disclosed is a process for the preparation of an interfacial enzyme immobilized on an insoluble support, by providing a bi-phase system comprised of an aqueous buffer solution and at least one first organic solvent; mixing said interfacial enzyme with the bi-phase system provided; adding the support to the obtained mixture and mixing; and isolating from the mixture obtained in the last step the interfacial enzyme immobilized on said support. The produced enzyme is locked in its catalytically active confirmation, and thus exhibits improved activity and stability. Also disclosed are uses of the produced enzymes, particularly in the preparation of biodiesel.

1 . A process for the preparation of an interfacial enzyme immobilized on an insoluble support, comprising the steps of:
 (a) providing a bi-phase system comprised of an aqueous buffer solution and at least one first organic solvent;   (b) mixing said interfacial enzyme with the bi-phase system provided in step (a);   (c) adding said support to the mixture of step (b) and mixing;   (d) isolating from the mixture obtained in step (c) the interfacial enzyme immobilized on said support.   
     
     
         2 . The process of  claim 1 , wherein prior to mixing with the enzyme biphasic solution obtained in step (b), said support is optionally washed to remove salts and organic materials, and is then treated with a surfactant dissolved in a second organic solvent. 
     
     
         3 . The process of  claim 1  or  claim 2 , wherein said insoluble support is capable of binding said enzyme by adsorption or by covalent binding to functional groups. 
     
     
         4 . The process of any one of  claims 1  to  3 , wherein said support is a porous support which may be organic or inorganic, preferably selected from the group consisting of porous inorganic support such as silica- or alumina-based supports, organic supports such as polymer-based support, wherein said support may optionally contain active functional groups such as epoxy or aldehyde groups, or ionic groups. 
     
     
         5 . The process of any one of  claims 1  to  4 , wherein said first organic solvent is selected from alkanes (such as octane), alcohols (such as n-octanol), aldehydes (such as decanaldehyde) and ketones (such as 2-octanone) and any mixture thereof. 
     
     
         6 . The process of any one of  claims 2  to  5 , wherein said surfactant is selected from sugar fatty acid esters, polyoxyethylene sugar fatty acid esters or ethers, medium- and long-chain alkyl glucosides, phospholipids, polyethylene glycol derivatives and quaternary ammonium salts. 
     
     
         7 . The process of any one of  claims 2  to  6 , wherein said second organic solvent is selected from alkanes, preferably such as n-hexane, ethers, preferably such diethyl ether, ketones, preferably acetone, and alcohols, preferably iso-propanol, and any mixture thereof. 
     
     
         8 . The process of any one of  claims 1  to  8 , wherein said enzyme is a lipase or a phospholipase. 
     
     
         9 . The process of  claim 8 , wherein said enzyme is selected from the group consisting of  Candida antarctica, Candida rugosa, Rhizomucor miehei, Pseudomonas  sp.,  Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Alcaligenes  sp.,  Burkholderia  sp.,  Thermomyces lanuginosa, Chromobacterium viscosum,  papaya seeds and pancreatin. 
     
     
         10 . An interfacial enzyme immobilized on a solid support, wherein said enzyme is locked in its catalytically active confirmation. 
     
     
         11 . An interfacial enzyme immobilized on a solid porous support, wherein said support is homogenously covered with a surfactant, preferably with a monolayer of said surfactant. 
     
     
         12 . The enzyme of  claim 10 , wherein said support is capable of binding said enzyme by adsorption or by covalent binding to functional groups. 
     
     
         13 . The enzyme of any one of  claims 10  to  12 , wherein said support is organic or inorganic, preferably selected from the group consisting of inorganic support such as silica- and alumina-based supports, organic supports such as polymer-based support, wherein said support may contain active functional groups such as epoxy or aldehyde groups and ionic groups or said support is an ion exchange resin. 
     
     
         14 . The enzyme of any one of  claims 11  to  13 , wherein said surfactant is selected from sugar fatty acid esters, polyoxyethylene sugar fatty acid esters or ethers, medium- and long-chain alkyl glucosides, phospholipids, polyethylene glycol derivatives and quaternary ammonium salts. 
     
     
         15 . The enzyme of any one of  claims 10  to  14 , being a lipase or a phospholipase. 
     
     
         16 . The enzyme of  claim 15 , wherein said enzyme is selected from the group consisting is selected from the group consisting of  Candida antarctica, Candida rugosa, Rhizomucor miehei, Pseudomonas  sp.,  Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Alcaligenes  sp.,  Burkholderia  sp.,  Thermomyces lanuginosa, Chromobacterium viscosum,  papaya seeds and pancreatin. 
     
     
         17 . An enzymatic process for the preparation of structured wax esters comprising the step of reacting a triglyceride source with an alcohol in the presence of an immobilized lipase as defined in  claim 15  or  16  or prepared by the process of  claim 9 , wherein said wax esters contain a surface-active component inherent to the raw starting material, whereby said wax esters possess improved water dispersibility 
     
     
         18 . The process of  claim 17 , wherein said alcohol is a C 2-22  alkanol, preferably cetyl alcohol. 
     
     
         19 . A process for the preparation of short-chain alkyl esters of fatty acids, preferably fatty acid methyl esters (biodiesel) comprising the step of:
 stepwise adding methanol to a plant, animal, algal or fish oil or a mixture of at least two of these oils that contain a lipase as defined in any one of  claims 10  to  16 , or prepared by the process of any one of  claims 1  to  9  and allowing the reaction to proceed under suitable conditions, until said oil triglycerides are converted to fatty acid methyl esters.   
     
     
         20 . The process of  claim 19 , wherein said plant oil is soybean, canola, rapeseed, olive, palm oil, sunflower oil, peanut oil, cotton seed oil, waste cooking oil or any oil triglycerides derived from inedible plant sources.