# US 11,999,977 B2 · In situ enzymatic degradation of hydrocarbon-polluted soils > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,999,977 B2 - Document ID: US11999977B2 - Status: Granted - Kind: B2 - Application number: 16/777,081 - Filed: 2020-01-30 - Published: 2020-07-30 - Granted: 2024-06-04 - Earliest claimed: 2019-01-30 - Assignees: Institut National De La Recherche Scientifique - Applicants: Institut National De La Recherche Scientifique - Inventors: Satinder Kaur Brar; Tayssir Guedri; Tarek Rouissi - Web page: https://patentgrep.com/patent/US11999977B2 ## CPC - C12N 9/0077: {with a reduced iron-sulfur protein as one donor (1.14.15)} - C02F 3/342: {characterised by the enzymes used} - C02F 3/344: {for digestion of mineral oil} - C12N 9/16: acting on ester bonds (3.1) - C12N 9/20: Triglyceride splitting, e.g. by means of lipase - C02F 2101/32: Hydrocarbons, e.g. oil - C12N 11/06: attached to the carrier via a bridging agent - C12N 11/18: Multi-enzyme systems - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes) ## Abstract The present invention provides crude enzyme extracts, cocktails and compositions from Alkanivorax borkumensis and methods for the enzymatic treatment and bioremediation of petroleum hydrocarbon polluted ecosystems. ## Claims ### Claim 1 A composition comprising at least one selected from the group consisting of: i) a cell free supernatant of Alkanivorax borkumensis (A. borkumensis) comprising an extracellular enzyme extract; ii) a supernatant of sonicated cells of A. borkumensis comprising an intracellular enzyme extract and an extracellular enzyme extract; and iii) combinations thereof; and between about 0.5% (v/v) and about 12% (v/v) of petroleum-derived hydrocarbons; wherein the composition comprises a plurality of polypeptides that exhibit at least two enzymatic activities selected from the group consisting of lipase activity, esterase activity, and alkane hydroxylase activity. ### Claim 2 (depends on 1) The composition of claim 1, wherein the plurality of polypeptides comprises one or more polypeptides that exhibit lipase activity. ### Claim 3 (depends on 1) The composition of claim 1, wherein the plurality of polypeptides comprises at least one polypeptide that exhibits alkane hydroxylase activity. ### Claim 4 (depends on 1) The composition of claim 1, wherein the A. borkumensis comprises Alakanivorax borkumensis strain SK2 (DSM 11573). ### Claim 5 (depends on 1) The composition of claim 1, wherein the A. borkumensis is grown in a culture medium comprising the petroleum-derived hydrocarbons. ### Claim 6 (depends on 1) The composition of claim 1, wherein the plurality of polypeptides exhibit lipase activity, esterase activity, and alkane hydroxylase activity. ### Claim 7 (depends on 6) The composition of claim 6, wherein the composition retains more than 70% of its original activity after one month when stored at low temperature between −20±1° C. and 4±1° C. ### Claim 8 (depends on 1) The composition of claim 1, wherein the plurality of polypeptides comprises at least one of a concentration of polypeptides exhibiting esterase activity from about 10 U/mL to about 500 U/mL, a concentration of lipase from about 10 to about 250 U/mL, and a concentration of alkane hydroxylase from about 1 U/mL to about 1000 U/mL. ### Claim 9 (depends on 1) The composition of claim 1, wherein the composition further comprises a biosurfactant produced from A. borkumensis and obtained from a culture supernatant thereof. ### Claim 10 (depends on 1) A method for treating a petroleum-hydrocarbon polluted-system, comprising the steps of: contacting the composition as defined in claim 1, with the petroleum-hydrocarbon polluted-system to form an inoculated medium; and incubating the inoculated medium for at least about 24 hours at a temperature of at least about 8° C. to achieve biodegradation of at least a portion of the petroleum-hydrocarbon. ### Claim 11 (depends on 10) The method of claim 10, wherein the petroleum-hydrocarbon comprises C10-C50 hydrocarbons, BTX, PAH, polychlorinated organic compounds, or mixtures thereof. ### Claim 12 (depends on 10) The method of claim 10, wherein the treating of the petroleum-hydrocarbon polluted-system comprises removing between about 30% and about 80% of total petroleum hydrocarbons (TPH). ### Claim 13 (depends on 1) A method for producing a composition as defined in claim 1, comprising the steps of: obtaining a crude culture medium comprising A. borkumensis cells grown on a medium comprising the about 0.5% (v/v) to about 12% (v/v) of petroleum-derived hydrocarbons; sonicating the crude culture medium to obtain a sonicated crude culture medium; and centrifuging the sonicated crude culture medium to recover the supernatant of sonicated cells. ### Claim 14 (depends on 13) The method of claim 13, further comprising incubating the cells under conditions comprising about 2% to about 10% of salt to achieve production of a biosurfactant. ### Claim 15 (depends on 14) The method of claim 14, wherein the medium comprises petroleum-derived hydrocarbons at a concentration of 3% (v/v), the petroleum-derived hydrocarbon being selected from the group consisting of hexane, hexadecane, motor oil, and mixtures thereof. ### Claim 16 (depends on 1) The composition of claim 1, wherein at least a portion of the polypeptides of the plurality of polypeptides is provided in admixture with chitosan. ### Claim 17 (depends on 16) The composition of claim 16, further comprising a polyanion to form nanoparticles with the chitosan. ### Claim 18 (depends on 17) The composition of claim 17, wherein the polyanion is tripolyphosphate (TPP) to form chitosan-TPP nanoparticles (CSNP). ### Claim 19 (depends on 18) The composition of claim 18, wherein the at least a portion of the polypeptides of the plurality of polypeptides exhibiting enzymatic activity is entrapped into the CSNP. ### Claim 20 (depends on 18) The composition of claim 18, comprising a polypeptide-to-CSNP ratio of between 1:1 to 7:1. ### Claim 21 (depends on 18) The composition of claim 18, wherein the at least a portion of polypeptides of the plurality of polypeptides exhibiting enzymatic activity comprises a polypeptide that exhibits alkane hydroxylase activity and a polypeptide that exhibits lipase activity, and wherein the polypeptide that exhibits alkane hydroxylase activity and the polypeptide that exhibits lipase activity are immobilized on the CSNP and exhibit more than a two-fold increase in in vitro half-life in comparison with free polypeptide. ### Claim 22 (depends on 1) The composition of claim 1, wherein the petroleum-derived hydrocarbons are selected from the group comprising hexane, hexadecane, motor oil, and mixtures thereof. ### Claim 23 (depends on 1) The composition of claim 1, further comprising a carrier, wherein the carrier comprises at least one selected from the group consisting of: sodium phosphate buffer and dimethyl sulfoxide.