# US 12,109,532 B2 · Variant of nitrous oxide reductase protein and method of reducing concentration of nitrous oxide in sample > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,109,532 B2 - Document ID: US12109532B2 - Status: Granted - Kind: B2 - Application number: 17/751,066 - Filed: 2022-05-23 - Published: 2023-06-08 - Granted: 2024-10-08 - Earliest claimed: 2021-12-08 - Assignees: Samsung Electronics Co., Ltd. - Applicants: Samsung Electronics Co., Ltd. - Inventors: Woo Yong Shim; Seung Hoon Song; Jae-Young Kim; Yu Kyung Jung - Web page: https://patentgrep.com/patent/US12109532B2 ## CPC - B01D 53/84: Biological processes - C12N 15/70: Vectors or expression systems specially adapted for E. coli - B01D 2251/95: Specific microorganisms - B01D 2255/804: Enzymatic - B01D 2257/404: Nitrogen oxides other than dinitrogen oxide - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Provided are recombinant microorganisms having a foreign gene encoding a variant of a nitrous oxide reductase protein, a composition comprising the recombinant microorganism or the variant of a nitrous oxide reductase protein for use in removing nitrous oxide in a sample, a variant of a nitrous oxide reductase protein, and a polynucleotide encoding the variant. ## Claims ### Claim 1 A recombinant microorganism comprising a foreign gene encoding a nitrous oxide reductase enzyme, wherein the enzyme is nitrous oxide reductase NosZ having at least 85% amino acid sequence identity to SEQ ID NO: 1, and wherein the enzyme comprises an amino acid alteration of an amino acid residue corresponding to position H78 of the amino acid sequence of SEQ ID NO: 1, wherein the microorganism belongs to the genus Escherichia. ### Claim 2 (depends on 1) The microorganism of claim 1, wherein the amino acid alteration is a substitution of the amino acid residue corresponding to position H78 with A, M, N, E, P, F, I, or C. ### Claim 3 (depends on 1) The microorganism of claim 1, wherein the amino acid alteration is an H78A, H78M, H78N, H78E, H78P, H78F, H78I or H78C substitution. ### Claim 4 (depends on 1) The microorganism of claim 1, further comprising a genetic modification that increases expression of an auxiliary gene product required for NosZ enzyme activity as compared to the expression of the auxiliary gene product in the microorganism without the modification, wherein the auxiliary gene product—is a NosR encoded by a nitrous oxide reductase R (nosR) gene, NosD encoded by a nitrous oxide reductase D (nosD) gene, a encoded by a nitrous oxide reductase F (nosF) gene, a NosY encoded by a nitrous oxide reductase Y (nosY) gene, and a flavinyltransferase ApbE encoded by an apbE gene, or a combination thereof. ### Claim 5 (depends on 1) The microorganism of claim 1, wherein the microorganism is Escherichia coli. ### Claim 6 A composition comprising a recombinant microorganism for reducing a concentration of nitrous oxide in a sample as compared to a sample without the recombinant microorganism, the recombinant microorganism comprising a foreign gene encoding a nitrous oxide reductase enzyme, wherein the enzyme is nitrous oxide reductase NosZ having at least a 85% amino acid sequence identity to SEQ ID NO: 1, and wherein the enzyme has an amino acid alteration of an amino acid residue corresponding to position H78 of the amino acid sequence of SEQ ID NO: 1, wherein the microorganism belongs to the genus Escherichia. ### Claim 7 (depends on 6) The composition of claim 6, wherein the amino acid alteration is a substitution of the amino acid residue corresponding to position H78 with A, M, N, E, P, F, I, or C. ### Claim 8 (depends on 6) The composition of claim 6, wherein the amino acid alteration is an H78A, H78M, H78N, H78E, H78P, H78F, H78I or H78C substitution. ### Claim 9 (depends on 6) The composition of claim 6, the microorganism further comprising a genetic modification that increases expression of an auxiliary gene product required for NosZ enzyme activity as compared to the expression of the auxiliary gene product without the genetic modification, wherein the auxiliary gene product is a NosR encoded by a nitrous oxide reductase R (nosR) gene, a NosD encoded by a nitrous oxide reductase D (nosD) gene, a NosF encoded by a nitrous oxide reductase F (nosF) gene, a NosY encoded by a nitrous oxide reductase Y (nosY) gene, and a flavinyltransferase ApbE encoded by an apbE gene. ### Claim 10 (depends on 6) The composition of claim 6, wherein the microorganism is Escherichia coli. ### Claim 11 A polynucleotide encoding a nitrous oxide reductase enzyme, wherein the enzyme is nitrous oxide reductase NosZ having at least 85% amino acid sequence identity to SEQ ID NO: 1, and wherein the enzyme comprises an amino acid alteration of the amino acid residue corresponding to position H78 of the amino acid sequence of SEQ ID NO: 1 and the amino acid alteration is a substitution of the amino acid residue corresponding to position H78 with A, M, N, E, P, F, I, or C. ### Claim 12 A method of reducing a concentration of nitrous oxide in a sample comprising nitrous oxide, the method comprising contacting a sample comprising nitrous oxide with a recombinant microorganism comprising a foreign gene encoding a nitrous oxide reductase protein, wherein the protein is nitrous oxide reductase NosZ having at least 85% amino acid sequence identity to SEQ ID NO:1, and—wherein the enzyme comprises an amino acid alteration of an amino acid residue corresponding to position H78 of the amino acid sequence of SEQ ID NO: 1 wherein the microorganism belongs to the genus Escherichia. ### Claim 13 (depends on 12) The method of claim 12, wherein the amino acid alteration is a substitution of the amino acid residue corresponding to position H78 with A, M, N, E, P, F, I, or C. ### Claim 14 (depends on 12) The method of claim 12, wherein the amino acid alteration is an H78A, H78M, H78N, H78E, H78P, H78F, H78I or H78C substitution. ### Claim 15 (depends on 12) The method of claim 12, wherein the microorganism further comprises a genetic modification that increases expression of an auxiliary gene product required for NosZ activity as compared to the expression of the auxiliary gene product in the microorganism without the modification, wherein the auxiliary gene product is a NosR encoded by a nitrous oxide reductase R (nosR) gene, a NosD encoded by a nitrous oxide reductase D (nosD) gene, a NosF encoded by a nitrous oxide reductase F (nosF) gene, a NosY encoded by a nitrous oxide reductase Y (nosY) gene, a flavinyltransferase ApbE encoded by an apbE gene, or a combination thereof. ### Claim 16 (depends on 12) The method of claim 12, wherein the microorganism is Escherichia coli. ### Claim 17 (depends on 12) The method of claim 12, wherein the contacting is performed in a closed container. ### Claim 18 (depends on 12) The method of claim 12, wherein the contacting comprises culturing or incubating the recombinant microorganism while contacting the recombinant microorganism with the sample comprising nitrous oxide. ### Claim 19 (depends on 12) The method of claim 12, wherein the sample is industrial wastewater or waste gas.