# US 7,611,835 B2 · Process for preparing multilayer enzyme coating on a fiber > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,611,835 B2 - Document ID: US7611835B2 - Status: Granted - Kind: B2 - Application number: 11/241,506 - Filed: 2005-09-30 - Published: 2007-04-05 - Granted: 2009-11-03 - Earliest claimed: 2005-09-30 - Assignees: Battelle Memorial Institute - Inventors: Jungbae Kim; Ja Hun Kwak; Jay W. Grate - Web page: https://patentgrep.com/patent/US7611835B2 ## CPC - C12Q 1/37: involving peptidase or proteinase - C12N 9/6427: {Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)} - C12N 11/082: obtained by reactions only involving carbon-to-carbon unsaturated bonds - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme “seeds” are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells. ## Claims ### Claim 1 A process for preparing a biocatalytic material, comprising the steps of: covalently attaching a plurality of preselected enzymes to an external surface of a preformed fiber, each of said preselected enzymes is covalently attached to a functional group on said surface of said fiber that secures said preselected enzymes to said surface; adding additional enzymes in the presence of a cross-linking reagent to said covalently attached preselected enzymes; and cross-linking said additional enzymes together to form aggregates of cross-linked enzymes that form a multilayer enzyme coating on said surface of said fiber that provide an enzyme activity greater than a monolayer of said preselected enzymes. ### Claim 2 (depends on 1) The process of claim 1, wherein said functional group is selected from the group consisting of: aminyl (—NH), sulfhydryl (—SH), alcohols (—OH), carbonyl (—C═O), carboxyl (—COOH), aldehydes (—CHO), isocyanates (—NCO), anhydrides, epoxides, silyl, and combinations thereof. ### Claim 3 (depends on 1) The process of claim 1, wherein said cross-linking reagent is selected from the group consisting of: a di-isocyanate, a di-anhydride, a di-epoxide, a di-aldehyde, a di-imide, 1-ethyl-3-dimethyl aminopropylcarbodjimide, and combinations thereof. ### Claim 4 (depends on 1) The process of claim 1, wherein said fiber is selected from the group consisting of: polymer fibers, co-polymer fibers, glass fibers, inorganic fibers, ceramic fibers, composite fibers, and combinations thereof. ### Claim 5 (depends on 1) The process of claim 1, wherein said fiber has a thickness between about 5 nm and about 30,000 nm. ### Claim 6 (depends on 1) The process of claim 1, wherein said fiber has a thickness greater than or equal to about 20 nm. ### Claim 7 (depends on 1) The process of claim 1, wherein said fiber has a length greater than or equal to about 1,000 nm. ### Claim 8 (depends on 1) The process of claim 1, wherein said enzyme activity has a longevity of greater than about 100 days without measurable loss in activity. ### Claim 9 (depends on 1) The process of claim 1, wherein said cross-linking reagent is selected from the group consisting of: 1-ethyl-3-dimethyl aminopropylcarbodiimide, an aldehyde, glutaraldehyde, a di-aldehyde; a di-imide; a di-isocyanate; an isocyanate; an anhydride; a di-anhydride; an epoxide; a di-epoxide; an alcohol, and combinations thereof. ### Claim 10 (depends on 1) The process of claim 1, wherein said biocatalytic material is used in a biocatalytic process or biocatalytic device. ### Claim 11 (depends on 1) The process of claim 1, wherein said biocatalytic material is used in a protein digestion process or protein digestion device. ### Claim 12 (depends on 1) The process of claim 1, wherein said biocatalytic material is used in a lab-on-a-chip process or a lab-on-a-chip device. ### Claim 13 (depends on 1) The process of claim 1, wherein said biocatalytic material is used in a bioremediation process or bioremediation device. ### Claim 14 (depends on 1) The process of claim 1, wherein said biocatalytic material is used in a biofuel cell process or biofuel cell device. ### Claim 15 (depends on 1) The process of claim 1, wherein said biocatalytic material is used in a detergent process or system. ### Claim 16 (depends on 1) The process of claim 1, wherein said biocatalytic material is used in a polymerase chain reaction process or polymerase chain reaction device. ### Claim 17 (depends on 1) The process of claim 1, wherein said biocatalytic material is used in a biosensor process or biosensor device.