United States Patent w
`Mulligan et al.
`
`[i i] Patent Number:
`[45] Date of Patent:
`
`5,037,758
`Aug. 6, 1991
`
`[75]
`
`[54] ENHANCED PRODUCTION OF
`BIOSURFACTANT THROUGH THE USE OF
`A MUTATED B SUBTTLIS STRAIN
`Inventors: Catherine N. Mulligan, Lachine;
`Terry Y-K Chow, Montreal, both of
`Canada
`[73] Assignee: Her Majesty the Queen in right of
`Canada, as represented by The
`National Research Council of Canada,
`Ottowa, Canada
`[21] Appl. No.: 296,215
`Jan. 11, 1989
`[22] Filed:
`[51] Int. CI.'
`[52] U.S. CI.
`
`C12N 1/20
`435/252.5; 435/172.1;
`435/839; 435/71.2
`435/172.1, 252.31, 252.5,
`435/839
`
`[58] Field of Search
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,687,926 8/1972 Arima et al
`
`435/839
`
`FOREIGN PATENT DOCUMENTS
`235461 11/1984 Fed. Rep. of Germany .
`2172898 10/1986 United Kingdom .
`
`OTHER PUBLICATIONS
`Cooper et al., "Enhanced Production of Surfactin from
`Bacillus subtilis By Continuous Product Removable and
`Metal Cation Additions," Applied and Environmental
`Microbiology, vol. 42 (3), pp. 408-412 (1981).
`Guerra-Santos et al., "Dependence of Pseudomonas
`aeruginosol Continuous Culture Biosurfactant Produc­
`tion on Nutritional and Environmental Factors", Ap­
`plied Microbiology and Biotechnology, vol. 24, pp.
`443-448 (1986).
`Primary Examiner—Douglas W. Robinson
`Assistant Examiner—Marian C. Knode
`Attorney, Agent, or Firm—Obion, Spivak, McClelland,
`Maier & Neustadt
`
`ABSTRACT
`[57]
`A B. subtilis strain possessing an enhanced surfactin
`production potential. The strain is a mutant of B. subtilis
`ATCC 21332 and has at least one mutation between
`Arg4 and HisAl sites of the genetic map of B. subtilis
`ATCC 21332. Also included in the present invention is
`B. subtilis strain having the identifying characteristics of
`ATCC 53813.
`
`3 Claims, No Drawings
`
`PETITIONERS
`
`EXHIBIT NO. 1029 Page 1 of 5
`
`

`
`1
`
`5,037,758
`
`ENHANCED PRODUCTION OF BIOSURFACTANT
`THROUGH THE USE OF A MUTATED B SUBTILIS
`STRAIN
`
`2
`posed so far are indirect methods having their limita­
`tions.
`Therefore, the obtention of a B. subtilis strain able to
`produce large quantities of surfactin would be highly
`5 desirable.
`
`BACKGROUND OF THE INVENTION
`SUMMARY OF THE INVENTION
`Biosurfactants are substances that have received a
`In accordance with the present invention, there is
`considerable amount of attention because of the fact
`provided a novel Bacillus subtilis strain whose genetic
`that they possess a wide variety of interesting proper­
`10 material has been modified through UV mutagenesis
`ties. For example, they can be used as oil recovery
`and which may be used for producing surfactin at levels
`agents, emulsifiers, antibiotics and antifungal agents. In
`which are higher than the levels encountered when
`fact, Arima et al. have demonstrated in 1968 Biochem.
`wild type Bacillus subtilis is employed. The genetically
`Biophys. Res. Commun. 31:488-494 that a biosurfactant
`modified strain of the present invention is a mutant of
`such as surfactin could reduce the surface tension of 15 Bacillus subtilis ATCC 21332 having at least one muta-
`water from 72 to 27 mN/m at a concentration as low as
`tion between Arg4 and HisAl sites of the genetic map
`0.005% and could also inhibit clot formation. Further­
`of B. subtilis ATCC 21332.
`more, Bernheimer and Avigad in 1970/ Gen. Microbiol.
`In fact, when biosurfactant production by the mutant
`61:361-369 have shown that surfactin could efficiently
`strain of the present invention is compared to that of the
`lyse erythrocytes while Hosono and Suzuki in 1983, J. 20 parent strain, it is found that the mutant strain produces
`Antibiot. 36:679-683 have demonstrated that bacterial
`as much as 3 to 4 times more biosurfactant than the
`parent strain in equivalent growth conditions over the
`spheroplasts and protoplasts as well as cyclic 3',5'-
`same period of time.
`monophosphate diesterase could also be inhibited by the
`Particularly, a subject mutant strain obtained through
`action of surfactin.
`Biosurfactants are produced as metabolic products or 25 U.V. radiation was deposited on Sept. 21, 1988 at the
`American Type Culture Collection, 12301 Parklawn
`membrane components. A considerable number of these
`Drive, Rockville, Md., 20852 and was given the ATCC
`compounds have been characterized and described by
`accession number 53813.
`various authors such Cooper et al. (1986, Microbiol. Sci.
`The mode of obtention as well as the utility of the
`3:145-149), Cooper and Zajic (1980, Adv. Appl. Mi­
`crobiol. 26:229-253), Margaritis et al. (1979, Biotech. 30 mutant strain of the present invention will be more
`Bioeng. 21:1151-1161), Rosenberg (1982, CRC Crit.
`readily illustrated by referring to the following descrip-
`Rev. Biotech. 1:109-132), Zajic and Steffens (1984, CRC
`t'on-
`Crit. Rev. Biotech. 1:87-107). These compounds are
`classified as lipopeptides, glycolipids, lipopolysaccha-
`rides, neutral lipids and fatty acids or phospholipids.
`They are surface-active due to their hydrophobic and
`hydrophilic regions. Since surfactants are used in many
`multiphase processes, they are very important industri­
`ally. Biosurfactants are potentially less toxic and more
`biodegradable than the synthetic compounds currently
`used. They can also be produced from a variety of sub-
`strates.
`In particular, lipopeptides are a very interesting class
`Obtention and selection of the mutant microorganism
`of compounds. Some examples such as amphomycin
`The purpose of the work that lead to the present
`(Bodanszky et al. 1973, J. Am. Chem. Soc. 95:2352-2357
`invention was to increase the yield in surfactin secretion
`and cyclosporin A (Dreyfuss et al., 1976, Eur. J. Appl.
`by Bacillus subtilis through genetic manipulations. Mu­
`Microbiol. 3:125-133; Riiegger et al. 1976, Helv. Chim.
`tation has been chosen since any change in the regula-
`Acta 59:1075-1092) are respectively known for their
`tory system of biosurfactant synthesis and secretion
`...
`.
`.
`,
`,
`antibiotic and antifungal activities. They contain both a J Q would result in an altered level of production,
`Thus, in order to obtain a mutant B. subtilis strain
`lipid portion and several amino acids.
`Bacillus subtilis ATCC 21332 produces surfactin.
`producing increased amounts of surfactin, Bacillus sub-
`Surfactin is a lipopeptide biosurfactant having as men-
`prototroph strain ATCC 21332 may be grown to
`tioned above quite interesting properties. Apart from
`logarithmic phase and then approximately 3000 cells are
`being a very powerful biosurfactant, surfactin has the 55 pjated on nutrient agar plates. The plates are then UV
`advantage of being easily isolated in pure form when
`radiated for 35 seconds with short wave in a Chromato-
`produced by microorganisms such as B. subtilis. How­
`Vue Cabinet Model CC-60 (UVP, Inc.). This dosage of
`ever, serious problems are associated with the industrial
`UV light had been previously determined to give a 10 to
`production of surfactin. Among these problems, the fact
`20% survival rate in the colonies. The UV-radiated
`that the yields are very low is certainly the most impor- 60 plates are then incubated at 37° C. in the dark until the
`tant one.
`colonies are visible.
`"
`Until now, the only methods which have been uti­
`In order to detect whether the obtained colonies
`lized to enhance production of surfactin by B. subtilis
`produce enhanced amounts of biosurfactant, the B.
`are strain selection or the manipulation of environmen­
`subtilis mutants derived from UV mutagenesis are rep-
`tal or nutritional factor such as described in Cooper et 65 lica plated or individually spotted onto rich medium
`al. in 1981, Appl. Environ. Microbiol. 42:408-412 and
`agar plates containing 5% sheep blood cells, 4% glu­
`Guerra-Santos et al. in 1986 Appl. Microbiol. Biotech.
`cose, 0.1% nutrient broth, 0.1% yeast extract and min­
`eral salts, as described by Cooper et al. in 1981 Appl.
`24:443-448. However, the methods that have been pro-
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The present invention relates to a novel bacterial
`strain of Bacillus subtilis obtained through genetic muta­
`tion by ultraviolet light of the Bacillus subtilis proto-
`troph strain ATCC 21332. The obtentiori of this strain
`40 allows for higher yields in the production of surfactin, a
`lipopeptidic biosurfactant possessing exceptional sur­
`face activity.
`
`45
`
`35
`
`PETITIONERS
`
`EXHIBIT NO. 1029 Page 2 of 5
`
`

`
`5,037,758
`
`4
`3
`may be further purified by redissolving in water (pH
`Environ. Microbiol. 42:408-412. These plates are then
`adjusted to 8.0 by the addition of NaOH), filtration
`screened for enhanced haemolytic activity by incuba­
`tion at 37° C. and by evaluation of the haemolytic zone
`through Whatman no. 1 paper, and re-extracting 3 times
`surrounding the colonies. It has been demonstrated by
`with the same solvent.
`Mulligan et al. in 1984, J. Ferm. Techno!. 62:311-314 5
`The amount of biosurfactant in the medium is deter-
`that the degree of lysis of red blood cells is related to the
`mined by amino acid analysis. In order to do so, a 10 ul
`level of surfactin production by B. subtilis.
`aliquot is dried and acid hydrolysed for 2.5 hours at 150°
`The mutant that produces a significantly larger ha­
`C. in a Waters PICO-TAG Amino Acid Analysis Sys­
`emolytic zone around the colony than the other sur­
`tem. The residue is then redissolved in 200 ul of sodium
`vived colonies in the parent strain is chosen. This mu- 10 buffer and injected on a Beckman System 6300 High
`tant is not an auxotroph as it grew on minimal media.
`Performance Analyser equipped with a Beckman
`Model 7000 Data Station. Analyses are performed ac­
`Determination of the location of the mutation
`cording to the general procedures described by Spack-
`The location of the mutation responsible for en­
`man et al. in 1958, Anal. Chem. 30:1190-1206. The ratio
`hanced surfactant production is determined through 15 Qf aspartic acid, glutamic acid, valine and leucine is
`protoplast fusion between the enhanced production
`found to be approximately 1:1:1:4 for the compounds
`mutant and BGSC strain 1A28 (ArgC4, HisAl and
`produced by each strain. This ratio is similar to the
`TrpC2). This fusion may be carried out according to the
`amino acid composition of surfactin shown by
`method by Akamatsu and Seguchi in 1987, Mol. Gen.
`Kakinuma et al. in 1969 Agric. Biol Chem. 33:1669-1671.
`Genet. 298:254-262. It was determined that either a 20
`Further confirmation of the structure of the biosur-
`single mutation or mutations clustered in a small region
`factants may be obtained by mass spectrometry. Based
`of DNA that acted as a unit may be responsible for the
`on the surfactin molecular formula (C53H93N7O13), the
`enhanced biosurfactant production of B. subtilis. The
`protonated molecular weight is 1036.6909. The spectra
`genetic mapping of the ATCC 53813 mutant with a
`of the compounds produced by the parent strain shows
`standard marker strain, B. subtilis IA28 demonstrated 25
`similar fragmentation patterns with respective M+ of
`that the mutation was located between ArgC4 and
`1036 and 1037. The mass spectra were obtained on a
`HisAl on the genetic map. Numerous mutations be­
`VG Analytical ZAB-SE double focussing mass spec­
`tween these two sites could also lead to an increased
`trometer. The accelerating voltage was 10 kV and the
`production of surfactant. .
`fast xenon atom beam was operated with an emission
`current of 1 mA at 8 kV. Mass spectra were recorded
`with the data, acquisition and calibration was performed
`with Csl.
`Surfactant production was compared to the produc­
`tion of surfactin by Bacillus subtilis ATCC 21332 under
`similar growth conditions. Results shown in Table 1
`demonstrate that the mutated strain of the present in­
`vention can produce at least 3 to 4 times more biosur­
`factant than the parent strain over the same time period.
`
`30
`
`Evaluation of the surfactin production of the mutant B.
`subtilis strain
`From a sheep blood agar plate, the B. subtilis mutant
`strain is inoculated into a 500 ml flask containing 100 ml
`of 4% glucose and mineral salts medium as described by 35
`in 1981, Appl. Environ. Microbiol.
`Cooper et al.
`42:408-412 supplemented with 3.2xlO_4M FeSCU.
`After 3 days of growth, 10 ml of the culture is trans­
`ferred to another similar flask. After 6 hours of growth,
`100 ml of this media may be used as an inoculum for a 40
`3.7 1 CHEMAP fermentor.
`The fermentor is operated under the following condi­
`tions: a 2.0 1 working volume, a temperature of 37° C,
`a 5.0 1/min aeration rate and pH control at 6.7. The
`surfactin concentrated in the foam is removed continu- 45
`ously into a flask on the air exhaust line as described by
`Cooper et al., in 1981, Appl. Environ. Microbiol.
`42:408-412.
`'
`Optical density is to be monitored at 600 nm through­
`out growth. Samples with optical densities above 1.0 50
`may be diluted to obtain a reading in the appropriate
`range. The readings are then multiplied by the dilution
`factor. Surface tension of the medium may be measured
`using a Fisher Surface Tensiomat Model 21 which em­
`ploys the du Noiiy method.
`•
`Surfactin may then be isolated by adding concen­
`trated HC1 to the broth after cell removal by centrifuga-
`tion as described by Cooper et al. in 1981, Appl. Environ.
`Microbiol. 42:408-412. The precipitated crude surfactin
`is then extracted 3 times with equal volumes of dichlo- 60
`romethane. This is followed by the removal of the sol­
`vent through evaporation under pressure. The surfactin
`
`TABLE 1
`Comparison of the growth and the amounts
`of biosurfactant produced by B. subtilis
`and the mutant strain of the present invention
`Amount of
`surfactin
`produced (mg)
`
`Growth after 40 hours
`(Optical density at 600 nm)
`
`Strain
`
`ATCC 21332
`ATCC 53813
`
`8.2
`8.3
`
`328
`1124
`
`We claim:
`1. A biologically pure culture of a Bacillus subtilis
`strain possessing an enhanced surfactant production
`potential and having at least one mutation between the
`55 Arg4 and HisAl sites of the genetic map of B. subtilis
`ATCC 21332.
`2. The biologically pure culture of B. subtilis strain of
`claim 1, wherein the mutation is obtained through ultra­
`violet light exposure.
`3. A biologically pure culture of Bacillus subtilis strain
`ATCC 53813.
`
`65
`
`PETITIONERS
`
`EXHIBIT NO. 1029 Page 3 of 5
`
`

`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`
`PATENT NO.
`
`: 5,037,758
`: AUGUST 6, 1991
`DATED
`INVENTOR(S) : CATHERINE N. MULLIGAN ET AL
`It is certified thai error appears in the above-ideniified patent and that said Letttrs Patent is hereby
`corrected as shown below:
`
`On the title page. Item [75]: Inventors: "Catherine N. Mulligan,
`Lachine, and Terry Y-K Chow,
`Montreal"
`Should read —Catherine N. Mulligan, Lachine, Terry Y-K Chow, Montreal,
`and Bernard Gibbs, Montreal—.
`
`Signed and Sealed this
`Eighth Day of March, 1994
`
`Attest:
`
`Attesting Officer
`
`Cnmmis'iioner of Patents and Trademarks
`
`BRUCE LEHMAN
`
`PETITIONERS
`
`EXHIBIT NO. 1029 Page 4 of 5
`
`

`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`
`PATENTNO. -.5,037,758
`: AUGUST 6, 1991
`DATED
`INVENTOR(S} : CATHERINE N. MULLIGAN ET AL
`It is certified that error appears in the abave-idtniified patent and that said Letttrs Patent is hereby
`unected as shown below:
`
`On the title page. Item [75]: Inventors: "Catherine N. Mulligan,
`Lachine, and Terry Y-K Chow,
`Montreal"
`Should read —Catherine N. Mulligan, Lachine, Terry Y-K Chow, Montreal,
`and Bernard Gibbs, Montreal—-
`
`This request was erroneously issued this request was denied on
`February 4, 1994.
`
`This request supersedes certificates of correction issued on
`March 8, 1994.
`
`Signed and Sealed this
`Twenty-sixth Day of April, 1994
`
`Attest:
`
`Attesting Officer
`
`Commissioner of Patents and Trademarks
`
`BRUCE LEHMAN
`
`PETITIONERS
`
`EXHIBIT NO. 1029 Page 5 of 5