# US 4,397,949 A · Preparation and immobilization of inulinase > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 4,397,949 A - Document ID: US4397949A - Status: Granted - Kind: A - Application number: 06/278,612 - Filed: 1981-06-29 - Granted: 1983-08-09 - Earliest claimed: 1980-06-27 - Assignees: Stamicarbon B.V. - Inventors: Peter J. H. Peters; Pieter L. Kerkhoofs - Web page: https://patentgrep.com/patent/US4397949A ## CPC - C12N 11/091: Phenol resins; Amino resins - C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)} - C12N 11/04: entrapped within the carrier, e.g. gel or hollow fibres - C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} - C12Y 302/01007: Inulinase (3.2.1.7) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12Y: ENZYMES ## Abstract Enzyme preparations are obtained from Aspergillus phoenicis having high inulinase activity, a very low Michaelis constant, thermal stability, optimum inulinase activity across a wide pH range, and little sensitivity to heavy metal ion inhibition. The enzyme may be immobilized on an organic macroporous polymer or in an alginate gel. ## Claims ### Claim 1 An inulinase-active enzyme preparation having insensitivity to heavy metal ion inhibition obtained by: (a) culturing an Aspergillus phoenicis mold culture for a period of several days under aerobic conditions in an aqueous medium containing a biologically assimilable nitrogen, carbon and phosphorus source and including inulin, at a pH of at least 4.0 up to about 7.0 and at a temperature of about 20.degree. C. to about 40.degree. C., and (b) recovering the inulinase-active enzyme solution thereby produced. ### Claim 2 (depends on 1) An enzyme preparation according to claim 1 wherein said culture medium is free of glucose. ### Claim 3 (depends on 1, 2) An enzyme preparation according to claim 1 or 2 wherein said mold culture is Aspergillus phoenicis CBS 294.80. ### Claim 4 (depends on 1, 2) An inulinase-active enzyme preparation according to claim 1 or 2 wherein said inulinase-active enzyme is classified as inulinase or inulase EC 3.2.1.7. ### Claim 5 An immobilized water-insoluble inulinase-active enzyme preparation having insensitivity to heavy metal ion inhibition obtained by: (a) culturing an Aspergillus phoenicis mold culture for a period of several days under aerobic conditions in an aqueous medium containing a biologically assimilable nitrogen, carbon and phosphorus source and including inulin at a pH of at least 4.0 up to about 7.0 and at a temperature of about 20.degree. C. to about 40.degree. C.; (b) thereafter recovering the inulinase-active enzyme solution which is thereby formed; (c) forming a reaction suspension of a resin in said enzyme solution, wherein said resin is obtained by reacting an organic macroporous polymer containing amino groups, hydroxyl groups or combinations thereof with glutardialdehyde, to adsorb said enzyme on said resin and thereby obtain an immobilized enzyme; and (d) thereafter recovering said immobilized enzyme from said suspension. ### Claim 6 An immobilized inulinase-active enzyme composition having insensitivity to heavy metal ion inhibition prepared by: (a) culturing an Aspergillus phoenicis mold culture for a period of several days under aerobic conditions in an aqueous medium containing a biologically assimilable nitrogen, carbon and phosphorus source and including inulin at a pH of at least 4.0 up to about 7.0 and at a temperature of about 20.degree. C. to about 40.degree. C.; (b) thereafter recovering the inulinase-active enzyme solution which is thereby formed; (c) admixing said enzyme solution with an alginate solution and gelling the alginate to produce an alginate gel containing the enzyme immobilized therein; and (d) thereafter recovering said immobilized enzyme composition which is thereby formed. ### Claim 7 (depends on 6) An enzyme composition according to claim 6, wherein said alginate solution is a calcium or sodium alginate solution. ### Claim 8 A process for obtaining an inulinase-active enzyme preparation having insensitivity to heavy metal ion inhibition by culturing a microorganism and recovering the inulinaseenzyme consisting essentially in the combination of steps of: (a) culturing an Aspergillus phoenicis mold culture for a period of several days under aerobic conditions in an aqueous medium containing a biologically assimilable nitrogen, carbon and phosphorus source and including inulin at a pH of at least 4.0 up to about 7.0 and at a temperature of about 20.degree. C. to about 40.degree. C.; and (b) thereafter recovering the inulinase-active enzyme solution which is thereby formed. ### Claim 9 (depends on 8) Process according to claim 8, wherein in step (a) said pH is between about 5 and about 7. ### Claim 10 (depends on 8, 9) Process according to claim 8 or 9, wherein in step (a) said temperature between about 25.degree. C. and 30.degree. C. ### Claim 11 (depends on 8) Process according to claim 8 wherein said recovery step (b) further includes concentrating said solution of inulinase-active enzyme by removing water. ### Claim 12 (depends on 8) Process according to claim 8, wherein in step (a), said pH is between 5.5 and 6.0. ### Claim 13 (depends on 8) Process according to claim 8, wherein said culture period is between 4 and 10 days. ### Claim 14 A process for obtaining an immobilized water-insoluble inulinase-activity enzyme composition having insensitivity to heavy metal ion inhibition, consisting essentially in the combination of steps of: (a) culturing an Aspergillus phoenicis mold culture for a period of several days under aerobic conditions in an aqueous medium containing a bioligically assimilable nitrogen, carbon and phosphorus source and including inulin at a pH of at least 4.0 up to about 7.0 and at a temperature of about 20.degree. C. to about 40.degree. C.; (b) thereafter recovering the inulinase-active enzyme solution which is thereby formed; (c) forming a reaction suspension of a resin in said enzyme solution, wherein said resin is obtained by reacting an organic macroporous polymer containing amino groups, hydroxyl groups or combinations thereof with glutardialdehyde, to adsorb said enzyme on said resin and thereby obtain an immobilized enzyme; and (d) thereafter recovering said immobilized enzyme from said suspension. ### Claim 15 In a process for hydrolyzing inulin with an immobilized inulinase-active enzyme preparation having insensitivity to heavy metal ion inhibition, the improvement consisting essentially in the combination of steps of: (a) culturing an Aspergillus phoenicis mold culture for a period of several days under aerobic conditions in an aqueous medium containing a biologically assimable nitrogen, carbon and phosphorus source and including inulin at a pH of at least 4.0 up to about 7.0 and at a temperature of about 20.degree. C. to about 40.degree. C.; and (b) recovering the inulinase-active enzyme solution which is thereby formed; (c) forming an immobilized water-insoluble inulinase-active enzyme preparation from said enzyme solution; (d) preparing an inulin containing solution; and (e) hydrolyzing the inulin in said inulin-containing solution with said immobilized enzyme preparation at a pH between 4 and 6 and at a temperature between 20.degree. C. and 65.degree. C. and at an inulin concentration in said inulin-containing solution of between about 5% to about 30% by weight based on the substrate solution. ### Claim 16 (depends on 15) In a process for hydrolyzing inulin according to claim 15, wherein said immobilization is effected by forming a reaction suspension of a resin in said enzyme solution, wherein said resin is obtained by reacting an organic macroporous polymer containing amino groups, hydroxyl groups or combinations thereof with glutardialdehyde, to adsorb said enzyme on said resin and thereby obtain an immobilized enzyme.