United States Patent us]
`Carrera et al.
`
`US005227294A
`[ii] Patent Number:
`[45] Date of Patent:
`
`5,227,294
`Jul. 13, 1993
`
`[75]
`
`[54] METHOD OF PRODUCING SURFACTIN
`WITH THE USE OF MUTANT OF BACILLUS
`SUBTILIS
`Inventors: Paolo Carrera; Paola Cosmina, both
`of Milan; Guido Grandi, Segrate, all
`of Italy
`[73] Assignee: Eniricerche S.p.A., Milan, Italy
`[21] Appl. No.: 718,464
`Jun. 20, 1991
`[22] Filed:
`[30]
`Foreign Application Priority Data
`20738 A/90
`Jun. 22, 1990 [IT]
`Italy
`22176 A/90
`Nov. 23, 1990 [IT]
`Italy
`C12P 21/02; C12P 1/125
`[51] Int. C1.5
`435/71.2; 435/71.1;
`[52] U.S. CI
`435/252.31; 435/252.5
`435/71.2, 71.1, 252.5,
`435/252.31
`
`[58] Field of Search
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,687,926 8/1972 Arima et al
`5,037,758 8/1991 Mulligan et al
`5,061,495 10/1991 Rossall
`
`435/71.2
`435/71.2
`435/70.1
`
`FOREIGN PATENT DOCUMENTS
`1803987 6/1969 Fed. Rep. of Germany .
`OTHER PUBLICATIONS
`Chemical Abstacts, vol. 112, No. 15, Apr. 9, 1990 ab­
`stract No. 137408Q, Schmidt, M. et al: "Influence of
`Manganese Ions on Growth and Surfactin Formation of
`Bacillus Subtilis", p. 582 & Dechema Biotechnol, Conf.
`1987 (publ. 1988), vol. 1; pp. 333-337.
`Applied Microbiology and Biotechnology, vol. 31, No.
`5/6, Oct. 1989, Springer Intematinal, pp. 486-489; Mul­
`ligan, C. et al.: "Enhanced Biosurfactant Production by
`a Mutant Bacillus Subtilis Strain".
`Primary Examiner—Herbert J. Lilling
`Attorney, Agent, or Firm—Sughrue, Mion, Zinn,
`Macpeak & Seas
`[57]
`ABSTRACT
`The present invention relates to a stable mutant of Bacil­
`lus subtilis which can produce surfactin with high
`yields, a method of producing surfactin with the use of
`the strain and the use of the surfactin obtained for phar­
`maceutical, energy and environmental problems.
`
`11 Claims, 11 Drawing Sheets
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 1 of 20
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`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 1 of 11
`
`5,227,294
`
`FIG. I
`
`1
`
`\
`
`C*
`
`mMm
`
`; ^
`
`issa.w-
`
`Dry weight / (g/l)
`
`FIG. 2
`
`o
`
`4
`
`3
`
`2
`
`0 -#
`0
`
`o9
`Q
`
`2 3 4 5 6 7 8 9 1 0
`Absorbance 600nm
`
`12 13 14 15 16
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 2 of 20
`
`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 2 of 11
`
`5,227,294
`
`FIG. 3
`
`Haemolysis diameter/(cm)
`
`•o
`
`1.4
`
`1.2
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0
`
`0
`
`2
`
`T
`4
`
`T
`8
`6
`10
`Surfactin / (mg/ml)
`
`12
`
`14
`
`16
`
`FIG. 4
`
`0.D.600
`
`©
`
`3
`
`25
`
`2
`
`1.5
`
`I
`
`0.5
`
`0
`0
`
`5
`
`10
`
`15
`time (h)
`
`20
`
`25
`
`30
`
`35
`
`—B-ATCC2I332
`
`-h ATCC55033
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 3 of 20
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`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 3 of 11
`
`5,227,294
`
`FIG. 5
`
`Optical density 600nm
`100 =
`
`Surfactin/ (g/l)
`
`* * * * * * IK
`
`*
`
`5
`
`• 4
`*
`
`10 i
`
`I =
`
`0-1 =
`
`0.01
`
`0
`
`10
`
`20
`
`30
`Time/ (h)
`
`40
`
`50
`
`0
`60
`
`O D " s r f
`
`FIG.6
`
`Optical density 600 nm
`
`20
`
`+-*
`
`*—*•
`
`+> * * • • • * » * *
`
`4
`
`1 5 -
`
`1 0 -
`
`0
`0 5
`
`T
`10
`
`T
`T
`T
`15 20 25 30 35 40 45 50 55 60
`Time/(h)
`-^Control
`Vol, Leu, lie -©-lie
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 4 of 20
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`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 4 of 11
`
`5,227,294
`
`FIG.7
`
`Surfactin /(g/l)
`
`5
`
`*
`
`-4
`
`* - 4
`
`0 5
`
`0
`10 15 20 25 30 35 40 45 50 55 60
`Time / (h)
`-— Control —*- Val,Leu,lle —
`
`FIG.8
`
`i ,
`
`3
`4
`
`5
`
`PHD
`
`• 6
`• ' t
`
`2
`i
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 5 of 20
`
`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 5 of 11
`
`5,227,294
`
`FIG.9
`
`Growth rate / (l/h)
`
`0 . 8 -
`
`0.6-
`
`0.4-
`
`0 . 2 -
`
`0 *
`0
`
`2.5
`
`5
`
`10
`7.5
`Time/(h)
`-©- 6 h Inoculum
`
`T
`12.5
`
`T
`15
`
`T
`17.5
`
`20
`
`20h Inoculum
`
`OD 600 nm
`100 F
`
`10 :
`
`0.1
`0
`
`10
`
`FIG. 10
`
`Relative amount
`
`20
`
`15
`
`- 10
`
`20
`
`1
`30
`Time /(h)
`Surfactin
`Cell density
`
`1
`40
`
`50
`
`0
`60
`
`PETITIONERS
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`EXHIBIT NO. 1016 Page 6 of 20
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`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 6 of 11
`
`5,227,294
`
`60
`
`- 50
`
`- 40
`
`- 30
`
`- 20
`
`- 10
`
`0
`100
`
`FIG. II
`
`OD 600 nm
`100 F
`
`Relative amount
`
`10 E
`
`0.1 !
`
`0.01
`0
`
`20
`
`40
`Time /(h)
`Cell density
`
`60
`
`80
`
`Surfactin
`
`FIG. 12
`
`o
`
`3
`4
`i
`
`6 t
`&
`
`3
`
`y
`2
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 7 of 20
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`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 7 of 11
`
`5,227,294
`
`FIG. 13a
`
`Optica! density
`100 3
`
`10 =
`
`0.1 =
`
`0.01
`0.0
`
`10.0
`
`20.0 30.0 40.0
`Time/(h)
`recycle —l— ultrafiltration
`
`i
`50.0 60.0 70.0
`
`FIG. I3b
`
`Surfactin / (g/l)
`
`TUBMB
`
`0
`
`10
`
`T
`20
`
`40
`30
`Time /(h)
`srf, ultrafil. ferm. -4- srf, recycle ferm.
`
`50
`
`60
`
`4
`
`3
`
`2
`
`0
`70
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 8 of 20
`
`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 8 of 11
`
`5,227,294
`
`FIG. 14
`
`Total peaks area (thousands)
`
`Q
`
`-Q-
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`5
`
`0
`
`0
`
`T
`0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
`Surfactin/(mg/ml)
`
`FIG. 15
`
`ro
`in in
`CM OJ
`
`ro
`CO
`00
`IO OJ
`
`N
`00
`N
`OJ
`
`i
`ro
`ro
`OJ
`
`s
`
`co
`OJ
`
`ro
`B
`
`oo
`ro
`OJ
`to O
`&>
`f0
`ro
`O
`sr
`
`h-
`CO
`ro
`
`N
`CVJ OJ
`a> co
`K CVJ
`
`00
`CVJ
`in
`(0
`CVJ
`10 gSj^S! ^
`in ro
`CVJ
`a>
`!D
`cb coooo ——
`
`co
`
`O
`
`ro
`i
`ro
`
`N-
`S2
`
`O
`OJ
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 9 of 20
`
`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 9 of 11
`
`5,227,294
`
`FIG.I6A
`
`3.3- r
`10- 1
`16.7-
`
`23.3-
`30-
`
`15.823
`19.71
`
`FIG.I6C
`
`3.3-
`10-
`
`16.7-
`
`23.3-
`
`30-
`
`13.767
`18.605
`> 22.165
`
`FIG.I6B
`
`3.3 -
`
`1 0 -
`
`16.7-
`
`23.3-
`
`30-
`
`FIG.I6D
`3.3-
`
`1 0 -
`16.7-
`
`23.3-
`30-
`
`12.742
`16.033
`16.662
`20052
`
`13.542
`18.453
`=> 22.023
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 10 of 20
`
`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 10 of 11
`
`5,227,294
`
`IOO-
`
`FIG. 17
`
`1022.7
`
`[H+H]+
`1000.8
`
`[H+H]+
`
`80-
`
`C320I igtRef.)
`912.4
`
`[H+H] +
`1036.8
`
`E+05
`
`60-
`
`40-
`
`20-
`
`0
`
`994.8
`992.8
`
`1038.8
`
`978.8
`964.8
`
`950.8
`936.8
`924.7
`
`T
`
`950
`
`VV
`
`1 r I 1
`
`1000
`
`C32ii20(Ref)
`1154.7 LL72-2
`1148.7
`1054.7
`/
`1084.8
`1126.7
`|I056.71 1112.8
`1
`
`N
`
`1186.7
`
`T
`
`1050
`
`! I [
`
`' ' »
`1100
`
`I
`
`T
`
`1150
`
`-H-O
`1200
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 11 of 20
`
`

`

`U.S. Patent
`
`July 13, 1993
`
`Sheet 11 of 11
`
`5,227,294
`
`FIG. 18
`
`3.300
`
`2856.6
`
`29588
`2929.9
`
`1734.0
`
`=> 1539.2
`
`1619.1
`
`1469.7
`~I386.8
`— 1261.4
`1207.4
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 12 of 20
`
`

`

`1
`
`5,227,294
`
`METHOD OF PRODUCING SURFACTIN WITH
`THE USE OF MUTANT OF BACILLUS SUBTJLIS
`
`5
`
`DESCRIPTION
`The present invention relates to a stable mutant of
`Bacillus subtilis which can produce surfactin with high
`yields, a method of producing surfactin with the use of
`the strain, and the use of the surfactin produced for
`pharmaceutical, energy and environmental problems.
`In recent years interest in biosurfactants of microbial
`origin for use as agents in the assisted recovery of hy­
`drocarbons, the stabilisation of emulsions and, more
`generally, in the energy and environmental fields has
`increased considerably since these products are biode­
`gradable and hence potentially less toxic than the syn­
`thetic compounds currently used.
`A biosurfactant of particular interest, which is pro­
`duced by Bacillus subtilis, is surfactin.
`This compound, which was characterised by
`Kakinuma et al. [Agric. Biol. Chem., 33: 971-972
`(1969); Agric. Biol. Chem., 33: 1523-1524 (1969); Agric.
`Biol. Chem., 33 973-976 (1969)], is a cyclic lipopeptide
`formed by a heptapeptide and a lipid portion constituted
`by a mixture of beta-hydroxy-fatty acids with chains
`having between 13 and 15 carbon atoms and has the
`following structure:
`
`L—Leu—beta—OH—Ci3_i5—L—Glu—L—Leu
`D*—Leu—L—Asp
`L—Val—D—Leu
`
`2
`However, this method has disadvantages resulting
`from the low yields of surfactin (0.05-0.1 g/litre of the
`crude product and 0.04-0.05 g/1 of the purified prod­
`uct).
`Consequently, this method is not very acceptable
`economically from a commercial point of view.
`Methods of improving surfactin yields have therefore
`been proposed in the art and these are based, essentially,
`on the use of particular culture media or mutants of the
`10 B.subtilis strain ATCC 21332 or particular technical
`solutions.
`Thus, for example, Cooper D.G. et al., [(1981), Appl.
`Environ. Microbiol., 42: 408-412)]describe a method of
`producing surfactin by the culture of B.subtilis ATCC
`15 21332 based, amongst other things, on the continuous
`removal of the foam which is produced during the fer­
`mentation and which contains 90-99% of the surfactin
`produced.
`The object of removing the foam is to prevent the
`20 inhibiting effect which high concentrations of surfactin
`have on bacterial growth.
`Under these conditions, however, a surfactin yield of
`0.7-0.8 g/litre is achieved.
`Moreover, the continuous removal of the foam may
`25 reduce the working volume of the fermentation medium
`which is discharged with the foam.
`Sheppard J. and Mulligan C., (1987), (Appl. Mi­
`crobiol. Biotechnol., 27: 110-116) describe a method by
`which a yield of 0.16 g/litre is obtained by the growth
`30 of B.subtilis cells in a medium supplemented with a
`hydrolysed peat protein.
`Moreover, Mulligan, C. et al [Appl. Microbiol. Bi­
`otech., 31: 486-489 (1989)]describe a method for im­
`Surfactin has the property of inhibiting the formation
`proving the yield of surfactin characterised by the use
`of blood clots and 3', 5' monophosphate diesterase and
`...
`.
`of lysing erythrocytes, spheroplasts and bacterial proto- ^ 0f a mutant of the B.subtilis wild-type strain ATCC
`plasts.
`21332.
`The authors report a surfactin yield of 0.562 g/litre
`Moreover, surfactin inhibits the fibrinogen-thrombin
`after 40 hours (p. 488, Table 1), that is, about 3.4 times
`reaction, thus slowing the formation of fibrin; this prop­
`greater than that obtained by the growth of the wild-
`erty makes the substance suitable as an active element
`for the preparation of compositions useful as anticoagu- ^ tVP6 strain under the same conditions,
`lants in the prophylaxis of thrombosis and for prevent-
`In sPite of the lal"ge amount of work carried out,
`ing diseases such as myocardial infarction, pulmonary
`therefore, no proposal has been found cheap enough to
`embolism, etc., in general.
`enable it to be developed on an industrial scale, mainly
`Surfactin shows anti-cholesterase activity, since it
`because of the low productivity of the micro-organisms
`lowers the levels of cholesterol in the plasma and in the 45 available hitherto,
`liver, as well as fungicidal and antibiotic activity.
`11 has now bee" found that the problems of the prior
`art can be overcome by a new mutant of B.subtilis
`The lipopeptide performs its bacteriostatic functions,
`for example, on the growth of mycobacteria, even at
`which can produce surfactin with high yields,
`low concentrations (5-10 ppm).
`Samples of this mutant strain were deposited at the
`Moreover, surfactin is a powerful surface-active 50 American Type Culture Collection on Apr. 23, 1990
`agent and in fact reduces the surface tension of water
`received the registration number ATCC 55033.
`from 72 mN/M to 27 mN/M at a concentration of
`A subject of the present invention is therefore the
`B.subtilis strain ATCC 55033.
`0.005%.
`A further subject of the present invention is a method
`Its multiple activity means that surfactin is of particu­
`lar interest, since it can be used widely in the pharma- 55 of producing surfactin with high yields including the
`use 0f the B.subtilis strain ATCC 55033.
`ceutical, energy and environmental fields.
`Another subject of the present invention is the use of
`Arima, K et al. (U.S. No. 3,687,926 and Biochem,
`the surfactin produced in the pharmaceutical, energy
`Bioph. Res. Commun., 31: 488-494 (1968)) describe a
`and environmental fields.
`method of perparing surfactin having the following
`Further subjects of the present invention will become
`structure:
`
`60
`
`(CH3)2CH(CH2)9CHCH2CO—L—Glu—L—Leu—D—Leu—L—Val
`I
`L—Leu—D—Leu—L—Asp
`
`O-
`
`characterised by the use of the B.subtilis strain ATCC
`21331 or ATCC 21332.
`
`clear from the following description and examples.
`
`PETITIONERS
`
`EXHIBIT NO. 1016 Page 13 of 20
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`

`

`5
`
`5,227,294
`4
`3
`In particular, the B.subtilis strain ATCC 55033 ac­
`The initial cell density of the fermentation is gener­
`ally equivalent to an O.D.eoo of between 0.025 and 0.040.
`cording to the present invention is characterised by
`Assimilable sources of carbon include carbohydrates
`good genetic stability (the ability to retain the mutation
`such as glucose, hydrolysed starches, molasses, sucrose
`acquired permanently) and good resistance to high con­
`or other conventional carbon sources.
`centrations of surfactin.
`Examples of nitrogen sources may be selected, for
`This strain was produced by the mutation of the
`example, from mineral ammonium salts such as ammo­
`B.subtilis wild-type strain ATCC 2i332, which is com­
`nium nitrate, ammonium sulphate, ammonium chloride
`monly available to the public. For this purpose, it is
`or ammonium carbonate, urea, or products containing
`possible to use conventional methods consisting of ex­
`posing cells of the wild-type strain to the action of 10 organic or inorganic nitrogen such as peptone, yeast
`extract or meat extract.
`chemical or physical mutagenic agents, selecting the
`The following cations and anions are also suitable for
`strains in which the surfactin yield is altered and, fi­
`the purposes of the present invention: potassium, so­
`nally, isolating those colonies in which productivity is
`dium, magnesium, iron, calcium, acid phosphates, sul-
`increased.
`Chemical mutagenic agents may be selected, for ex-
`phates, chlorides, manganese and nitrates.
`According to the present invention a fermentation
`ample, from diethyl sulphate, NMU (nitrosomethyl
`medium having the composition given in Example 2 is
`urethane), NMG (N-methyl-N'-nitro-N-nitrosoguani-
`preferred.
`dine), and physical agents may be selected from X-rays,
`The fermentation is carried out in a vessel (a fer-
`UV-rays (ultra-violet) and gamma rays in mutagenic
`20 menter or bioreactor) with stirring and intensive aera­
`doses.
`tion, at a temperature usually between 25° and 40° C.,
`According to one embodiment of the present inven­
`preferably between 30° and 37° C., with the continuous
`tion, the B.subtilis wild-type strain ATCC 21332 was
`removal of the foam formed which contains more than
`mutagenised with the use of NMG in concentrations
`98% of the surfactin produced.
`such as to induce mutations in the genomes of the mi­
`25 Quantities of sterile compressed air which may vary
`cro-organisms.
`from 0.2 to 1.0 vol/vol/minute are diffused into the
`The mutants which could produce surfactin with
`fermentation medium.
`high yields (the overproducer mutants) were then se­
`The pH of the fermentation medium is kept between
`lected by analysing the sizes of the haemolysis haloes
`6.0 and 7.2 and preferably between 6.7 and 6.9.
`which appeared around the B.subtilis colonies grown on
`30 The pH may be adjusted, for example, by the addition
`a culture medium such as, for example, TBAB
`of a basic aqueous solution such as an aqueous solution
`(DIFCO) to which blood had been added.
`of ammonia, potassium hydroxide, sodium hydroxide,
`In fact, it is known that the diameter of the haemol­
`sodium carbonate or potassium carbonate.
`ysis halo is proportional to the quantity of surfactin
`It is preferable, however, to keep the pH to the de-
`produced by the cells of B.subtilis (Mulligan, C. and 35 sjre(j value with the use of 10N sodium hydroxide
`Cooper, D. (1984), J. Ferment. Technol., 62: 158-179).
`(NaOH)
`The overproductivity of the B.subtilis mutant ATCC
`The stirring speed, which is one of the reasons for the
`55033 thus isolated was then confirmed by the fermenta­
`production of the foam, is generally selected so as to
`tion of the strain in question in a flask with the use of the
`allow high bacterial growth without having dramatic
`wild-type strain as a control.
`40 effects on the formation of the foam.
`B.subtilis ATCC 55033 seems to be particularly suit­
`The initial stirring speed is typically between 150 and
`able for producing surfactin with high yields by a fer-
`400 rpm, preferably between 200 and 300 rpm.
`mentation method.
`Finally, the working volume of the fermentation
`A method according to the invention may consist, for
`which appears to be a critical element as regards the
`example, of preparing an inoculum of the mutagenised 45 formation of the foam, is selected so as to limit the
`strain under aerobic conditions in an aqueous medium
`production of foam and hence the loss of the cellular
`containing assimilable sources of carbon and nitrogen.
`biomass which is discharged with the foam produced.
`The medium is kept under agitation at a temperature
`The removal of the foam formed during the fermenta-
`between 25° and 40° C., preferably from 30° to 37° C.
`tion process reduces the working volume and results in
`for a period shorter than 20 hours, preferably from 6 to 50 the loss of the cells contained therein.
`10 hours.
`There are two possible approaches to overcome this
`In fact, it has been found that an "old" inoculum (20
`problem. In one case, fresh sterile culture medium may
`hours) causes foam production which is difficult to
`be added to the bioreactor continuously (continuous
`control to the point that after 5 hours the fermentation
`fermentation) and, in the second case, the culture broth
`has to be stopped because of the extensive loss of the 55 .(containing the bacterial cells) which is discharged
`culture medium which is discharged with the foam.
`with the foam may be recycled into the bioreactor (the
`"Young" inocula (6-10 hours) on the other hand
`recycling of the culture medium).
`enable the foam to form gradually with a limited loss of
`According to a preferred embodiment of the method
`the culture medium.
`of the present invention, the culture medium which is
`A percentage of the inoculum of between 5% and 60 eliminated with the foam removed is recycled to the
`10% (volume/volume) of the working volume is then
`bioreactor with the use, for example, of an automatic
`added to the fermentation medium which contains as-
`system as shown in FIG. 8.
`similable sources of carbon and nitrogen as well as vari­
`Such a fermentation could present problems due to
`ous cations, anions and possibly vitamins such as biotin
`the fact that the concentration of the surfactin in the
`or thyamine and an aminoacid suitable for encouraging 65 recycled medium could have an adverse effect on the
`cell growth and the production of surfactin, selected
`further production of surfactin in the bioreactor because
`from L-Valine, L-Leucine, D-Leucine and L-Isoleu-
`of the inhibiting effect of the substance on bacterial
`cine.
`growth (Cooper et al.).
`
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`25
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`30
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`5
`Surprisingly, however, the results obtained have
`BRIEF DESCRIPTION OF THE DRAWINGS
`shown an increase in the biomass and constant surfactin
`values in the bioreactor.
`FIG. 1
`A photograph of a plate of TBAB (DIFCO) medium
`This indicates that the supply of a concentrated solu­
`tion of surfactin from the collection container to the 5 to which blood has been added, showing the haemolysis
`haloes of the B.subtilis wild-type strain ATCC 21332
`bioreactor has a limited effect on cell growth and that
`and of the B.subtilis overproducer mutant ATCC 55033.
`the surfactin has no inhibitory effect on its own synthe-
`sis by the mutant B.subtilis strain ATCC 55033.
`FIG. 2
`These results suggest that, unlike the B.subtilis wild-
`A calibration curve in which the weight (g/1) of the
`type strain ATCC 21332, the mutant of the present 10 solid biomass is shown on the ordinate and the absor-
`bance at 600 nm is given on the abscissa.
`invention may be more resistant to high concentrations
`of surfactin.
`FIG. 3
`A curve correlating the diameter (cm) of the haemol-
`According to a further embodiment of the method of
`ysis halo produced on TBAB medium to which blood
`the present invention, the foam and the culture medium
`removed from the bioreactor may be treated by an 15 has been added (on the ordinate) with the quantity of
`ultrafiltration system assembled as shown in FIG. 12.
`surfactin (mg/ml) added to the medium (on the ab-
`The culture medium is then recycled to the bioreactor
`scissa).
`FIG. 4
`without the surfactin.
`A growth curve for the B.subtilis mutant ATCC
`When the fermentation is carried out under the pre­
`55033 showing time sis a function of the absorbance
`ferred conditions, a concentration of crude surfactin of
`measured at a wavelength of 600 nm and compared
`from 2.0 to 4.0 g/litre is obtained within a period of
`with the growth curve of the wild-type strain.
`from 40 to 90 hours.
`The time is expressed in hours on the abscissa and the
`At the end of the fermentation process, the surfactin
`absorbance (O.D.eoo) on the ordinate.
`is recovered from the foam and from the a cellular or
`FIG. 5
`cellular supernatant liquid and purified.
`A graph showing the cell growth and surfactin pro­
`Conventional methods may be used for this purpose,
`duction detected during the fermentation of the B.subti­
`such as, for example, precipitation by an inorganic acid
`lis mutant ATCC 55033 in a flask.
`such as sulphuric or hydrochloric acid or by a com­
`The time is shown in hours on the abscissa; the absor­
`pound of a bivalent metal such as calcium, or by satura­
`bance value of the cell culture at a wavelength of 600
`tion with ammonium sulphate.
`nm is shown (on a logarithmic scale) on the ordinate on
`This treatment enables the selective precipitation of
`the left-hand side, and the quantity (g/1) of surfactin
`the surfactin and of some lipopeptides and lipoproteins
`produced by B.subtilis ATCC 55033 is shown on the
`produced by B.subtilis.
`right-hand side.
`This precipitation step may be preceded by the treat- 35
`FIG. 6
`ment of the a cellular supernatant liquid by an ultrafil­
`tration system in order to remove coarser impurities and
`A graph showing the cell growth detected during the
`fermentation of the B.subtilis mutant ATCC 55033 in a
`concentrate the working volume to be purified.
`flask in the presence of the aminoacids Val, Leu and He
`The precipitate containing the surfactin is then puri­
`fied with the use of one of the known techniques such 4Q (*) and of He (o).
`as, for example, extraction with organic solvents such as
`The time is shown in hours on the abscissa; the absor­
`bance of the cell culture at a wavelength of 600 nm is
`chloroform or methylene chloride or saline precipita­
`shown (on a logarithmic scale) on the ordinate on the
`tion by CaCh or NaCl.
`The method of the present invention produces quanti­
`left-hand side.
`FIG. 7
`ties of from 1.2 to 2.0 g/litre of purified surfactin (99%). 45
`A graph showing the surfactin production during the
`The chemical-physical characterisation of the surfac­
`fermentation of the B.subtilis mutant ATCC 55033 in a
`tin produced by the method of the present invention
`may be carried out by conventional methods with the
`flask in the presence of the aminoacids Val -fLeu +Ile
`(*) and He (o).
`use of chromatographic, spectroscopic or spectrometric
`techniques.
`50 The time is shown in hours on the abscissa; the quan­
`tity (g/1) of surfactin produced is given on the ordinate
`The results obtained by mass spectrometry (FAB),
`on the right-hand side.
`infrared spectrometry (IR), nuclear magnetic resonance
`(NMR) and high-pressure
`liquid chromatography
`FIG. 8
`(HPLC) confirmed the data in the literature.
`A diagram showing a 2-litre fermenter with recycling
`Moreover, the presence of fatty-acid molecules 55 in which:
`1) is the fermenter, 2) is an air-inlet line, 3) is a pump,
`which differ not only in the length of their carbon
`4) is a used-air line, 5) is a foam-collection container and
`chains (C13-C15) but also in their structures which may
`6) is the line for recycling the culture medium.
`be normal, iso or anteiso was confirmed.
`The characterisation of the surface-active and aggre­
`FIG. 9
`This drawing shows the rate of growth of the B.subti­
`gative properties of the surfactin produced by the 60
`lis strain ATCC 55033 achieved in a 2-litre fermenter
`method of the present invention confirmed the data in
`with the use of inocula of different ages.
`the literature.
`The surfactin obtained by the method according to
`The time is shown in hours on the abscissa; the
`growth rate is shown on the ordinate.
`the present invention is therefore particularly suitable as
`FIGS. 10 and 11
`a surface-active agent, as a stabilising agent for thera- 65
`Graphs showing the cell growth and surfactin pro­
`peutic compounds such as medication for the treatment
`duction detected during the fermentation of the B.subti­
`of thromboses, embolisms and inflammation, and in the
`lis mutant ATCC 55033 "with recycling".
`energy and environmental fields.
`
`PETITIONERS
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`5,227,294
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`8
`sporulation medium having the following composition:
`
`Nutrient broth (DIFCO)
`KC1
`MgS04
`Agar (DIFCO)
`MnCl2.4H20
`FeS04.7H20
`Na2S04
`H2O
`PH
`
`8.0 g/1
`1.0 g/1
`1.25 X 10-' g/1
`16.0 g/1
`1.98 X 10-3 g/1
`2.78 X lO"4 g/1
`1.42 X 10-1 g/1
`liter
`
`7.0
`
`7
`The time is shown in hours on the abscissa; the absor-
`bance value of the cell culture at a wavelength of 600
`nm is shown (on a logarithmic scale) on the ordinate on
`the left-hand side, and the corresponding quantity de­
`tected by the measurement of the areas of the three 5
`main chromatographic peaks in which the presence of
`surfactin was shown is indicated on the right-hand side.
`FIG. 12
`A diagram showing the production of surfactin with
`the continuous ultrafiltration of the product, in which:
`1) is the fermenter, 2) is an air-inlet line, 3) is a pump, 4)
`is a used-air line, 5) is a foam-collection container, 6) is
`an ultrafiltration cartridge and 7) is a container for the
`A loop of the preculture was then used to inoculate
`recycled culture medium.
`15 10 ml of DIFCO VY medium (Veal Infusion Broth 25
`FIG. 13
`g/1 and yeast extract 5 g/1) and grown at 37° C. for 16
`A) shows comparative curves of the growth of the
`hours.
`B.subtilis mutant ATCC 55033 during fermentation
`A portion (100 fj,l) of the culture was transferred into
`with recycling and with ultrafiltration. The time is
`a 100 ml flask containing 10 ml of fresh VY medium and
`shown in hours on the abscissa; the absorbance value of
`grown with gentle stirring (200 revolutions per minute,
`rpm) at 37° C. until an optical density (O.D.) of 0.7
`the cell culture at a wavelength of 600 nm is given (on
`determined at 600 nm by a Beckman spectrophotometer
`a logarithmic scale) on the ordinate.
`(mod. DU70) was achieved.
`B) compares the quantities of surfactin present in the
`The bacterial cells were then separated from the
`bioreactor in a fermentation test with ultrafiltration and
`supernatant liquid by centrifuging at 5,000 rpm for 10
`in a fermentation test with recycling.
`minutes with the use of a Mod. SS. 34 rotor on a Sorvall
`The time is shown in hours on the abscissa; the quan­
`supercentrifuge, washed with 5 ml of TM buffer (Tris-
`tity of surfactin (g/1) is given on the ordinate.
`HC1 0.05M, maleic acid 0.05M, (NRvhSCU 1 g/litre,
`FIG. 14
`MgS04.7H20, 0.1 g/litre, Ca(N03)2 5 mg/litre, Fe-
`A calibration curve correlating the quantity of surfac-
`,
`tin expressed in g/1 (abscissa) with the resulting value 30 SO4.7H2OO.25 mg/litre) brought to pH 6.0 with 5N
`NaOH, separated again by centrifuging in the manner
`of the sum of the areas of six peaks produced by HPLC
`described above, and resuspended in 5 ml of TM buffer.
`analysis (ordinate).
`5 ml of a solution of hydrated N-methyl-N'-nitro-N-
`FIG. 15
`nitrosoguanidine (1:1 with H2O) (Fluka) in TM buffer
`A chromatographic profile of a 25 fig sample of puri­
`(0.3 mg/ml) was then added to the cellular suspension
`fied surfactin. The three main peaks are constituted by 35
`and then incubated with stirring at 37° C.
`surfactin bound to fatty acids with lengths varying be­
`After 30 minutes the suspension was centrifuged
`tween 13 and 15 carbon atoms.
`again and the cells recovered were washed with 5 ml of
`FIG. 16
`TM buffer and then resuspended in 50 ml of fresh VY
`The chromatographic profiles of this drawing are the
`medium.
`result of the direct analysis of 50 p.1 of the acellular 40
`Portions (1 ml) of the suspension were grown with
`supernatant liquid withdrawn during fermentation. The
`gentle stirring at 37° C. for one night and, after the
`profiles relate to samples taken 3, 4, 27 and 30 hours
`addition of 0.2 ml of sterile glycerol, were frozen at
`after the start of the experiment. The presence of the
`-80° C.
`product is shown clearly by the appearance and subse­
`The B.subtilis mutants were then selected by the
`quent development of the chromatographic peaks relat- 45
`spreading of serial dilutions (about 2X ^cells/plate) of
`ing to the various components of surfactin (C13-C14-
`the portions on TBAB medium plates (Tryptose Blood
`C15)
`Agar Base (DIFCO) 33 g/litre) to which 5% of defibri-
`FIG. 17
`nated ram's blood (SCLAVO S.p.A.,) filtered through
`The characterisation of a sample of purified surfactin
`sterile gauze had been added, after sterilisation at 120°
`by the FAB (Fast Atom Bombardment) technique. The 50
`C. for 20 minutes.
`main peaks shown relate to products having molecular
`After thermostatically-controlled Incubation at 37° C.
`weights of 1008, 1022 and 1036 respectively, and corre­
`for 16-24 hours, the diameter of the haemolysis haloes
`sponding to surfactin fractions bound to beta-fatty acids
`which appeared around the bacterial colonies were
`with chains of various lengths.
`determined (FIG. 1).
`FIG. 18
`In fact, it is known that the size of the haemolysis halo
`An IR (infrared ray) spectrum of a sample of purified
`is proportional to the quantity of surfactin produced by
`surfactin. The interpretation of the spectrum is given in
`the B.subtilis cells (Mulligan, C. and Cooper, D. (1984),
`Example 6C.
`J. Ferment. Technol., 62: 158 -179).
`The present invention will be described further by
`One of the surfactin-overproducer colonies, desig-
`60 nated B.subtilis SMS 274, was deposited as ATCC
`the following examples.
`55033.
`EXAMPLE 1
`Preparation of the B.subtilis mutant which is an
`overproducer of surfactin
`A preculture of the B.subtilis wild-type strain ATCC
`21332 (available from the American Type Culture Col­
`lection) was grown at 37° C. for one night on Schaeffer
`
`EXAMPLE 2
`Production of surfactin in a flask
`A colony of B.subtilis SMS 274 was used to inoculate
`10 ml of VY medium and grown at 200 rpm, at 37° C.
`for 16 hours.
`
`10
`
`20
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`25
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`55
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`65
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`Glucose
`NH4CI
`KH2PO4
`NaHP04
`MgS04.7H20
`CaClz-ZH:©
`FeS04.7H20
`MnCl2.4H20
`EDTA
`.
`pH
`
`40.00 g/1
`4.00 g/1
`4.00 g/1
`5.64 g/I
`0.20 g/1
`1.00 X 10-3 g/I
`20.00 X 10- 3 g/1
`1.98 X 10-4 g/1
`1.50 X ID"3 g/1
`7.0
`
`5
`
`30
`
`50
`
`55
`
`9
`100 jul of this preculture were then transferred into a
`flask with a capacity of 2 litres containing 1 litre of
`minimum medium having the following composition:
`
`10
`EXAMPLE 3
`Investigation of the effect of aminoacids on the growth
`and production of surfactin by B.subtilis SMS 274
`A series of tests was carried out to check the effects
`of aminoacids on the growth and production of surfac­
`tin by the mutant SMS 274.
`The tests were carried out as in Example 2 in 2-litre
`jo flasks each containing 1 litre of minimum medium sup­
`plemented with 5 mg/1 of each of the following
`aminoacids: L-Leucine (Leu), L-Valine (Val), L-Isoleu-
`cine (He) and D-Leucine.
`The results showed that the presence of the three
`15 aminoacids L-Leu, L-Val and L-Ile in the fermentation
`The culture was carried out with stirring (250 rpm) at
`medium induced an increase in the biomass (FIG. 6).
`37° C. for 24 and 48 hours in a New Brunswick thermo­
`During the first 20 hours, the surfactin production
`was similar to that obtained in the control (the same
`statically-controlled incubator.
`In all the experiments the B.subtilis strain ATCC
`strain grown in a medium without additional aminoa-
`21332 grown under the same conditions was used as a 20 cids), however, the surfactin content in the medium
`increased during the stationary phase and at the end of
`control.
`the fermentation the quantity of surfactin was about
`Cell growth (biomass) was monitored by the determi­
`30% greater than that found in the control (FIG. 7) and
`nation of the optical density of the culture broth at 600
`Table 1.
`nm (DU 70 spectrophotometer, Beckman Instruments,
`25 When only He was added to the fermentation me­
`Inc., USA) with the use of dishes with optical paths of
`dium, it was observed that cell growth was inhibited
`1 cm (Bio-Rad Laboratores, USA). The O.D. values
`and there was a delay of 1 ho