Abstract:
Addition of a water-soluble polyelectrolyte (i.e. an anionic or cationic polymer) significantly increases the hydrolysis rate of esters in the presence of lipases. The invention provides a process for hydrolysis of water-insoluble ester in the presence of a lipase characterized by the presence of a water-soluble polyelectrolyte. The invention also provides a method of increasing the rate of hydrolysis of water-insoluble ester in the presence of a lipase by incorporation of a water-soluble polyelectrolyte.

Description:
This application is a continuation application of application Ser. No. 08/122,435, filed Sep. 24, 1993, now abandoned, the contents of which are incorporated herein by reference and a continuation of PCT/DK92/00137 filed on Apr. 30, 1992. 
    
    
     TECHNICAL FIELD 
     This invention relates to a process for hydrolysis of water-insoluble ester in the presence of a lipase, particularly to such a process for hydrolysis of pitch (resin) in pulp, and to a method of increasing the rate of hydrolysis of water-insoluble ester in the presence of a lipase by incorporation of a polyelectrolyte. 
     BACKGROUND ART 
     It is known that lipases can be used with advantage for efficient hydrolysis of water-insoluble esters, particularly triglycerides (e.g. JP-A 51-080305, JP-A 58-126794, JP-A 59-210893, GB-A 2,176,480, WO 88/02775). 
     It is also known that some types of pulp made from wood have a high pitch content, e.g. various types of mechanical pulp. This can cause so-called pitch troubles in papermaking such as paper contamination or paper breaks. Pitch contains considerable amounts of triglycerides, more commonly known as fats, and other esters. 
     It is the object of this invention to provide an improved process for ester hydrolysis, applicable to hydrolysis of resin esters. 
     STATEMENT OF THE INVENTION 
     We have found that, surprisingly, addition of a water-soluble polyelectrolyte (i.e. an anionic or cationic polymer) significantly increases rate of esters in the presence of lipases. 
     Various metal cations have been reported to affect lipase activity, and cationic surfactant has been reported inhibit lipase activity (Nishio et al., Agric. Biol. Chem., 51 (1), 181-186, 1987; C. E. Ibrahim et al., Agric. Biol. Chem., 51 (1), 37-45, 1987). The effect of polyelectrolytes on lipase activity has not been described. 
     Accordingly, the invention provides a process for hydrolysis of water-insoluble ester in the presence of a lipase, characterized by the presence of a water-insoluble polyelectrolyte. The invention also provides a method of increasing the rate of hydrolysis of water-insoluble ester in the presence of a lipase by incorporation of a water-soluble polyelectrolyte. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Polyelectrolyte 
     The polyelectrolyte used in the invention may be any water-soluble polymer that contains functional groups which ionize in water. It may be cationic or anionic. A group of preferred anionics is anionic polyacrylamide, e.g. a copolymer of acrylamide and acrylate (such as sodium acrylate). 
     Some preferred cationic polymers are those contaning tertiary or quaternary amine groups. An example is cationic starch having diethylamino-ethyl groups or 2-hydroxy,2-(trimethylamino-methyl)ethyl groups attached to the hydroxyl group in the 6-position of the repeating glucose unit of the starch molecule. 
     Another example is cationic polyacrylamide, e.g. a copolymer of acrylamide with N-(dimethyl-amino-methyl)-acrylamide, dimethyl-amino-ethyl methacrylate or trimethyl-amino-ethyl methacrylate. A further example is cationic polyamine such as quaternary polyamine and polyethyleneimine. 
     Use of the above-mentioned polyelectrolytes is particularly advantageous in papermaking where these polymers may simultaneously act flocculants or retention aids. 
     The amount of polyelectrolyte is preferably 2-1000 ppm, preferably 10-200 ppm in the reaction mixture, or 0.1-10 kg/ton of dry matter, particularly 0.3-3 kg/t. 
     Lipase 
     For reasons of economy, microbial lipases are preferred. Examples of suitable enzymes are lipases derived from strains of Pseudomonas (especially Ps. cepacia, Ps. fluorescens, Ps. fragi and Ps. stutzeri), Candida (especially C. antarctica (e.g. lipase A or B, see WO 88/02775) and C. cylindracea), Humicola (especially H. brevispora, H. lanuginosa, H. brevis var. thermoidea and H. insolens), Chromobacterium (especially C. viscosum) and Aspergillus (especially A. niger). 
     The amount of lipase will typically correspond to a lipase activity of 1,000-100,000 LU/kg dry matter or 50-5,000 LU/litre (LU=Lipase Unit, defined in WO 89/04361). 
     Ester Hydrolysis Process 
     Typical process conditions are pH 3-7.5, particularly 4-7, a temperature from ambient to 80° C., particularly 30°-60° C., and reaction times of 0.5-3 hours. 
     The process of the invention can be used for any lipase-catalyzed hydrolysis of water-insoluble esters, particularly triglycerides. 
     Thus, the process of the invention may be used for fat hydrolysis in the production of fatty acids, glycerides and/or glycerol from fat or oil. The ester may be a liquid at ambient temperature, such as soy bean oil and many other oils, or it may be a high melting fat, such as beef tallow. 
     Hydrolysis of Resin Esters 
     The process of the invention is particularly applicable to the hydrolysis of resin esters during a pulping or paper-making process, e.g. to avoid pitch troubles such as paper contamination, paper breaks or contamination of process equipment. 
     The process of the invention may be applied to any pitch-containing pulp, especially to pulps with a considerable content of triglycerides and other esters from pitch. Examples are pulps produced by mechanical pulping, alone or combined with a gentle chemical treatment, such as GW (Ground Wood), TMP (Thermo Mechanical Pulp) and CTMP (Chemical Thermo Mechanical Pulp). 
     Hydrolysis of esters in pitch according to the invention can be done in the pulping or stock preparation section, where addition of polyelectrolytes is particularly advantageous since it can also act as a retention or flocculation aid. The pulp typically has a consistency of 0.2-5% dry substance. 
    
    
     EXAMPLES 
     Example 1 
     Red pine (Pinus radiata) ground wood pulp was treated with Humicola lipase in the presence of various polyelectrolytes. After the reaction the degree of triglyceride hydrolysis was determined by quantitative TLC using latroscan™. 
     Conditions were: 4% pulp slurry, pH 4.5, temperature 40° C., agitation 300 rpm. The dosage of polyelectrolyte and enzyme is given below as ppm/DS. Results: 
     
         ______________________________________                  Dosage   Relative Amount       Dosage     of       of Trigly-       of poly.   Lipase   cerides (*)Polyelectrolyte       (ppm/DS)   (ppm/DS) (%)______________________________________None (control)         0        1000     100Anionic, High       1000       1000     79MolecularPolyacrylamide-copolymerCationic, High       1000       1000     67MolecularPolyacrylamide-copolymerStrongly Cationic,       1000       1000     64High MolecularPolyacrylamide-copolymerQuaternary Polyamine       1000       1000     67Cationic Polymer       1000       1000     71______________________________________ (*): Determined by quantitative TLC; Iatroscan Method. 
    
     It is seen that all the anionic and cationic polymers tested increased the hydrolysis of triglyceride. 
     Example 2 
     To verify the effect of polyelectrolytes on lipase activity another experiment was done, using two different cationic polymers. Conditions were: 4% pulp slurry, pH 4.5, temperature 40° C., 2 hours reaction time, agitation 300 rpm. Dosage of polyelectrolytes and enzyme are given below as ppm/DS. 
     
         ______________________________________Dosage (ppm/DS)       Dosage   Relativeof                    (ppm/DS) amountCationic   Quarternary   of       TriglyceridesPolyner Polyamine     Lipase   (%)______________________________________0       0             0        1000       0             1000     451000    0             1000     361000    0             0        1000       1000          1000     320       1000          0        100______________________________________ (*): Determined by quantitative TLC; Iatroscan Method.