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Publication numberUS3840433 A
Publication typeGrant
Publication dateOct 8, 1974
Filing dateMay 9, 1972
Priority dateSep 23, 1968
Publication numberUS 3840433 A, US 3840433A, US-A-3840433, US3840433 A, US3840433A
InventorsAndressen Otto, Aunstrup Knud, Outtrup Helle
Original AssigneeNovo Terapeutisk Labor As
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Dehairing of leather
US 3840433 A
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Description  (OCR text may contain errors)

United States Patent [191 Aunstrup et al.

DEHAIRING OF LEATHER Inventors: Knud Aunstrup, Farum; Otto Andressen, Copenhagen; Helle Outtrup, Varlose, all of Denmark Assignee: Nono Terapeutisk Laboratorium A/S, Bagsvaerd, Denmark Notice: The portion of the term of this patent subsequent to July 4, 1989, has been disclaimed.

Filed: May 9, 1972 Appl. No.: 251,646

Related US. Application Data Continuation-in-part of Ser. Nos. 761,546, Sept. 23, 1968, Pat. No. 3,723,250, and Ser. No. 772,830, Nov., 1968, Pat. No. 3,674,643.

US. Cl. 195/6 Int. Cl C146 1/06, C12b 1/00 Field of Search 195/6, 65, 62, 66 R [451 *Oct. 8, 1974 Primary ExaminerLione1 M. Shapiro Attorney, Agent, or FirmFidelman, Wolffe, Leitner & Hiney [5 7] ABSTRACT Dehairing of skins and hides in an alkaline medium pH of about 12.5, such as by lime treatment, is accelerated by inclusion of certain alkali resistant proteolytic enzymes. Preferred embodiments are the proteases from Bacillus alcalophilus, and from the Bacillus sp. strains NCIB 10313, 10317, 10147.

6 Claims, 2 Drawing Figures Pat. No. 3,723,250 and Ser. No. 772,830

DEHAIRING' OF LEATHER.

This application is a continuation-in-part of prior applications Ser. No. 761,546 filed Sept. 23, 1968 now filed Nov. 1, 1968 now Pat. No. 3,674,643.

This invention relates to an enzymatic procedure for dehairing hides and skins and more particularly relates to dehairing of skins and hides by treatment at a pH range of -13.0 with a new class of proteolytic enzymes.

A well known conventional step in the conversion of skins and hides'into leather is the liming step. The skin or hide is treated with a saturated solution of hydrated lime containing sodium sulfide or a similar chemical which liberates the sulfide ion. After a period of from 12 hours to 6 days, the hair can be readily scraped off. Frequently activators or sharpening agents like amines,

' cyanides, etc. are added to hasten the process, e.g. to

a 24-hour treatment. Sometimes agents are added in sufficient quantity to destroy much, sometimes all of the hair. The actual liming procedure will vary in details from animal to animal, and from tannery to tannery.

Other techniques for dehairing, or unhairing as it is sometimes called, include treatment with enzymes, including proteases. Unfortunately, previously available proteolytic enzymes are not effective at high pH, and particularly at pH about 12.5, the pH of saturated lime solutions. Liming is widely believed to do more than loosen hair. The high pH of liming seems to loosen the fiber weave and swell the collagen fibers. Moreover, the skin or hide takes up water, and becomes more amenable to subsequent treatment procedures. Accordingly, dehairing by enzymatic action has, heretofore, been inconsistent with liming, requiring a separate step. Liming remains a principal treatment procedure in the preparation of leather, despite the substantial pollution problems involved with the waste effluents of the liming pits. The leather industry has considered itself committed to a liming step, and its choice between the more time consuming (e.g. 1 week) treatment with lime alone or lime and sulfide followed by mechanical removalof the hair, and possible sale of the hair as a byproduct, and a quicker treatment with lime plus an additive which acts to destroy the hair. Speed is desirable but costly. There is loss of the hair byproduct, the existence of a more severe pollution problem from the effluent, and the expense of the additive.

According to the present invention, the lime treatment is made faster by incorporating one or more of the alkali resistant protease disclosed in the aforementioned copending applications Ser. No. 761,546 and Ser.v No. 772,830. Thus practice of the invention involves incorporating one or more of these proteolytic enzymes of the serine type showing optimal proteolytic activity against hemoglobin at apH above 9 when measured by the Anson method. Such proteolytic enzymes are produced by aerobic cultivation of species of Bacillus in a nutrient medium comprising assimilable carbon and nitrogen sources, using protease-forming species of Bacillus isolated from nature on a nutrient medium having a pH value within the range of 9 to l 1, or mutants thereof and maintaining the pH value of the production nutrient medium between 7.5 and 10.5 during the main part of the cultivation. An advantageous group of proteolytic enzymes which may be incorporated are those showing -100% of maximum activity when measured at pH 12 by the Anson method. A readily identified group of proteolytic enzymes which maybe incorporated are the proteolytic enzymes obtained by the cultivationof Bacillus alcalophilus. All these enzymes are capable of withstanding the highly alkaline pH of a saturated lime solution or a soda ash solution of equivalent pH.

The enzyme acts to speed up dehairing considerably without, at the same time, destroying the hair. Moreover, presence of the enzyme seems to be beneficial otherwise. indications exist that the area of leather yield may be slightly increased. In addition, bating may be faster and require less enzyme.

The proteolytic enzymes available heretofore to the art have degraded too rapidly at pH 12. Most proteases are not very effective in alkaline media. indeed development of detergent enzymes capable of effective enzymatic action in the pH range of 8-9, was a considerable achievement, but their effectiveness at still higher pH is inadequate. At best the concept of combined liming and enzymatic dehairing steps constituted an impractical dream. The practice of the present invention involves heretofore unknown proteolytic enzyme preparations.

As has been described in Ser. No. 761,546, there exists in nature a great number of hitherto unknown bacteria that forms during their metabolism, proteolytic enzymes which display optimal proteolytic activity against hemoglobin at high pH-values up to 10 to 12, and which have other properties making them excellently suited for use within different industrial fields,

including notably deharing.

From samples of soil, animal manure and a number of other sources in nature the inventors have isolated about one hundred strains of bacteria, carried out taxonomic investigation and found that all of the hitherto unknown bacteria belong to the genus Bacillus, but that none of them belonged to any species known to the inventors, that, to the best of the inventors knowledge,

they did not belong to the same species. Furthermore,

within the same species there were in most cases different strains and several varieties.

For the purpose of isolating the hitherto unknown bacteria referred to above, use has been made of a novel technique which is characterized by the fact that the isolalation is effected on nutrient media having a pH-value within the range of 9 to l l, and intended for detection of production of proteolytic enzymes.

In other words, the samples of soil, animal manure or other sources from nature have been spread on nutrient media having the high pH-value referred to and the bacteria able to grow under such alkaline conditions are then isolated and subjected to further investigations as to species and enzyme production. in most case, use

proteolytic enzyme production in view, has been carried out both in shake flasks and in tanks in pilot plants with artificial aeration. The yields obtained have been determined by the well-known Anson hemoglobin method, cfr. Journal of General Physiology, 22, 79-89 I (l9 59). One Anson unit means throughout this specification the amount of proteolytic enzyme digesting hemoglobin at pH-v'alue of 10.1 and a temperature of 25C during a reaction time of minutes with such an initial velocity that per minute there is formed such an amount of split products which cannot be precipitated with trichloroacetic acid that these split products give the same'colour with phenol reagent as does one milliequivalent of tyrosine.

The process for producing the proteolytic enzymes belongs to the known art in which the enzymes are produced by aerobic cultivation of bacteria in a nutrient medium containing assimilable carbon and nitrogen sources, and the characteristic feature regarding the present enzymes consists in maintaining a pH-value of the nutrient medium within the range of 7-12 during the cultivation, using species of the genus Bacillus able to grow and produce the proteolytic enzymes within the'pH-rangereferred to, and'recovering from the medium the proteolytic enzymes formed during the cultivation. 1

Experiments seem to show that it is convenient to carry out the cultivation at a pH-value of the culture medium within therange of 7.5 to 10.5.

The nutrient medium is composed in agreement with the principles of the known art.

For further details regarding exemplary strains and screening and cultivation details, reference is again made to copending application Ser. No. 761,546 filed Septf23, 1968. The enzymes of particular interest'for deharing purposes are those showing 80-1 00% of maximum activity when measured atpH 12 by the Anson method. A preferred embodiment of the invention is use of the proteolytic enzymes available from the strains of NCIB-10147; NCIB-10813 and NCIB-10317.

hoek", 1, 141-147 (1934), A. Vedder described his experiments' to isolate certain bacteria from faeces. By enrichment in alkaline peptone water and cultivation on what he called glycocoll plates" containing hemoglobin, KOH, glycocoll and peptone-agar, or better carbonate plates containing hemoglobin, K CO KHCO and peptone agar, Vedder succeeded in isolating 16' strains of a bacterium belonging to the genus Bacillus, and being considered by Vedder to belong to a novel species which he called Bacillus alcalophilus because this species did only grow at pH values above 7, preferably within the range of 8.6 .to 10. The species Bacillus alcalophilus was incorporated in the 6th edition of Bergeys Manual of Determinative Bacteriology, but not in the 7th edition.

One strain of Bacillus alcalophilus was deposited with National Collection of Type Cultures in London under the NCTC number 4553 and with National Collection of Industrial Bacteria in Edinburghunder the NCIB number 8772, where the strain by a mistake had been them'useful for use in dehairing at high pH.

The Bacillus .alcalophilus is cultivated under submerged aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen sources, maintaining the pH value of the nutrient medium between 7.5 and 1 1, and the proteolytic enzyme produced extracellularly during the cultivation is recovered.

The nutrient medium is composed in agreement with the principles of the known art. Suitable assimilable carbon sources are carbohydrates, such as saccharose, glucose, starch, cereal grains, malt, rice, sorghum, etc. The carbohydrate concentration may vary within rather wide-limits, e.g. up to 25% and down to l5%, but usually 8-10% would be suitable, the percentage being calculated as dextrose. It has been found that the presence in the'nutrient medium of carbohydrates will give rise to the formation of acidic components, resulting in a decrease of the pH value during thecultivation. As it is essential to maintain a pH value of the nutrient medium within the range of 7.5 to 11, preferably 8 to 10, during the cultivation, measurements should be taken that the pH value does not fall below that range for any essential period during the Cultivation. In order to keep the pH value within the required range, a limited amount of carbohydrates may be used together with a buffer substance which is able to maintain the required pH value. Carbonates, and particularly sesquicarbonates, used in a concentration of'up to 0.2 M in the medium are able to create a pH value of about 9.3 to 10.5 respectively.

Also other buffer systems, such as phosphate buffers may be used.

It is'also possible to initiate the cultivation with a low carbohydrate content and to add small amounts of carbohydrates successively during the cultivation.

A third possibility is to make use of automatic pH control by addition of various basic-reacting substances used in this art.

The use of carbonates and 'sesquicarbonates as pH controlling substances is very useful and it is surprising that it is possible during the cultivation on an industrial scale to use these compounds in the concentrations referred to.

The nitrogen source in the nutrient medium may be of inorganic and/or organic nature. Suitable inorganic nitrogen sources are nitrates and ammonium salts, and among the organic nitrogen sources there are quite a number known for use in fermentation processes and in the cultivation of bacteria. Illustrating examples are soybean meal, cotton seed meal, peanut meal, casein, corn steep liquor, yeast extracts, urea, albumine, etc.

trace substances.

1 The temperature at which the cultivation takes place is normally within the same range as in the known cultivation of known species of the genus Bacillus. Usually a temperature between 25 and 40C. is convenient. The

. temperature is preferably about 30 to 37C.

As the cultivation has to be carried out under aerobic conditions, it is, when using fermentation tanks, necessary to make use of artificial aeration. The amount of air is similar to that used in the known cultivation processes. In general, maximum yields of the proteolytic enzymes will be obtained after a cultivation time of 1 to 5 days.

"For cultivation with proteolytic enzyme production and recovery in view, use has been made of the deposited strain from both of the collections referred to above, viz. strain NCTC 4553 and NClB 8772. These strains show optimal growth at a pH value within the range of 7.8 and 9.0. The cultivation has been carried out both in shake flasks and in pilot plant fermentors with artificial aeration. The yields obtained have been determined by the well-known Anson hemoglobin method, cfr. Journal of General Physiology, 22, 79- 89 (1939). One Anson unit means throughout this specification the amount of proteolytic enzyme digesting hemoglobin at a pH value of 10.1 and a temperature of 25C. during a reaction time of 10 minutes with such an initial velocity that per minutethere is formed an amount of split products which cannot be precipitated with trichloracetic acid that these split products give the same color with phenol reagent as does one milliequivalent of tyrosine.

For cultivation the following two media were used:

1) Medium BPFA with the following composition:

Potato flour 50 g per liter of ta water Saccharose 50 g do. Barley flour 50 g do. Soybean meal g do. Sodium caseinate l0 g do.

2) Medium BSX with the following composition:

Barley flour I 100 g per liter of tap water Soybean meal 30 g The starch in the media was liquefied with alpha amylase before sterilization. Both these media were adjusted to the desired pH value by the addition of sesquicarbonate or soda under sterile conditions.

The experiments in shake flasks were carried out in 500 ml shake flasks, each of the flasks containing 100 ml of the nutrient medium BPFA and BSX, respectively, which were sterilized beforehand by autoclaving 220 revolutions per minute, and the temperature during the cultivation was 30C.

. 6 The maximum proteolytic activity measured in Anson units per kg of the nutrient medium appears in Table 1 below.

Table l Bacillus Maximum proteolytic Final pH alcalophi- Nutrient activity value lus medium Anson units per kg I NClB 8772 BPFA l4 7.7 25 7.7 BSX 6 8.8 .5 8.2

NCTC 4553 BPFA 16 7.9 l3 7.0

By fractionatedprecipitation with ethyl alcohol enzyme preparations in powder form were prepared as it appears from the following Table ll.

Anson units in total 7:

The strain NClB 8772 has also been cultivated in 550 liters stainless steel tanks under submerged conditions and artificial aeration while using 250 liters of the nutrient medium BPFA referred to above. The pH value of the medium was adjusted on 10.2 by sterile addition of 15 liters of 2 M soda solution. The temperature during the cultivation was 30C., the velocity of the agitator 570 rpm and the aeration 0.25 m air per minute. After a cultivation period of 110 hours the pH value of the cultivation broth was 8.9 and the proteolytic activity was 17 Anson units per kg, measured at pH 10. l.

The enzyme preparations produced by the processes described above have been tested in regard to'the proteolytic activity against hemoglobin at different pH values and different temperatures.

The activity was measured at pH 7, 7.5, 8, 9, l0, l1 and 12 at 25C., and expressed in percentage of maximum activity. The results are compiled in Table Ill below.

The activity was also measured at a constant pH value of 10.1 and varing temperatures, viz. 25, 37, 50, 60, 66 and of Celsius. The results of these measurements are compiled in Table IV below.

Temperature, C.

8 I The enzyme C-303 (NCIB 10147) was tested against a standard detergent proteolytic enzyme i.e. Alcalase,

for initial activity at 259C. at various pH levels, and for residual activity after 24 hours at various pH levels.

result 3' l l 2 2 37 66 75 5 The substrate was denatured hemoglobin. The results NCTC 4553 may be seen in the hereto attached drawing wherein:

% activity 7 FIG. 1 shows the effectof pH on activity; and PH 14 32 65 26 2 FIG. 2 shows the pH stability of the same two enzymes. As ma be seen in FIG. 1 the H o timum for the In geperal enzyme preparzltlons i forPracnce preferred dehairing enzyme is 9 but tl ie enzyme is alof the lnvention consist of a SOllCl or l1qu1d m1xture of most q y active up to P 12 activity of the protgolytc enzylme-s and o-ther components; th-ey erence' enzyme drops off rapidly with pH to an appar- Zg gi i f igz fiz g ai gfgg z lg ent 60% residual activity at pH 12, but this level of acrp g 15 tivity is transitory. As may be seen in FIG. 2, essentially or Substances .avmg other than enzymauq zictwlty'use' all the activity of the reference enzyme has been deful for the unhty of the gnzyme cclmposltlonsT when stroyed by 24 hours at pH in excess of about pH 1 1.5. the enzymes 3" not .used {n.cryStallm'e f they may The enzyme C-303 (NClB-l0147) still retains approxibe i l g by g g i gl such mately 60% of its initial activity after 24 hours at pH 325: .3 1 2: 3: z g g g g g g gi gi 12. The pH stability curves of FIG. 2 demonstrate the y incompatibility of the prior art proteolytic enzymes The nzyme compoltlons P lquld form corisiltute with the liming procedure. Although the pH stability solutlon or suspenslonswhich may contain stabilizers, tests were not conducted under dehairing circum If necessary: stances, the pH test results are consistent with dehair- The alkali stable enzyme preparations or composi- 25 ing experience Hons for dehalrlng use normally q an enzyme con' I To repeat, tlieleather art is committed to liming. A tent not exceedmg about 10% by welght' In cases combination liming and enzyme treatment can be car- 9 the enzyme conlem may be .zonslderably ried out effectively with the alkali stable enzymes. hlgher The fqnowmg expnmems made 78 preferred For further understanding of the present invention, enzymes are Intended to lllustrat? utlhty of the the following specific examples thereof are presented. kaline stable enzymes for dehairing purposes under A employ the enzyme 0303 high pH.

A salted cowhide (the butt) is sliced in pieces mea- Example I Surmg about 20 X 4 The pleces are steeped 24 Cowhides soaked according to conventional procehimrs a fatand are scraped. The places of dures were drummed for 3 hours, then for 5 minutes are plac.ed m 400 ml different enzyme each hour for 24 hours under the following conditions: lutlons contained 1n glasses havmg a volume of 500 ml. 1 The glasses are incubated at 30C. for 24 hours. The pieces are then removed from the solutions and the HM ratio. 200% hairs are scraped off wlth a piece of plexiglass. The de- 40 Temperature: 25 -27c. hairing effect is evaluated in'accordance with the fol- Enzyme wncenmmm g'gfif Mum: lowing scale: Lime concentration: 4% (on'wet salted hide wt) 1. Easy and completeremoval of the hairs I 2. Easy removal of the hairs, but spots of hairs re- The hair was completely loose in 24 hours. After submained on the hide sequent processing, the finished leather was identical in 3. No or difficult removal of the hairs physical properties to a set of lime-sulfide treatment The proteolytic enzyme solutions-used contain 1 g calcontrols but had slightly greater area yield. cium hydroxide per 130 g of water. The amount ofen- E l H zymes appears from the below Table V, also indicating xamp e the pH values at the beginning and the end of the de- Cowhideswere drummed intermittently for 24 hours hairing process together with the results thereof. W underthe following conditions:

Table'V Enzyme from control C 303 C 367 C 372 strain NClB 10147 NCIB 10313 NCIB 10317 Amount of enzyme 0 0.5 5 0.5 5 0.5 5 Anson units Initial pH value 11.9 11.9 11.9 11.9 11.8 12.0 11.9

Final pH value 11.9 11.8 11.3 11.8 1L8 11.9 ll.8

Dehairing Float ratio: 250% Temperature: 23-25%. Enzyme concentration: 0. l7: (wt'71 per volume of float) Lime concentration: 271 (on wet salted hide wt) Sodium sulfite con.: V/r (do) The hair was loose in 24 hours. The hides were processed in conventional manner but without bating. They were slightly firmer but otherwise comparable with lime-sulfide controls. In a similar test employing 0.05% enzymes concentration the unhairing was not as satisfactory.

Example lll Cowhides were drummed intermittently for 24 hours using the following conditions, after which period the hair was loose.

Float ratio: l5071 Temperature: C.

Enzyme concentration: 0.075% (wt% per volume of float) Lime concentration: 47r (on wet salted hide wt) The finished leather was comparable in all respects to chrome tanned, lime-sulfide controls. After dyeing,

the enzyme treated hides were somewhat darker on both-sides than the controls. However, some of the fine hairs of the enzyme treated hides did not appear to have been loosened.

Example IV Cowhides were drummed intermittently in sodium carbonate/bicarbonate buffers under the following conditions:

Float ratio: (i007:

Temperature: 30C.

Enzyme concentration: 0.17! (wt71 per volume of float) pH: l0.0

Hair loosening was completed in 24 hours. The leather was then chrome tanned. The enzyme treated leather was comparable in all respects to lime-sulfide controls. V

I Example V 54-kgs of hide were treated according to a drum painting technique under the following conditions:

0.171 (on the wet salted wt) 47: (on the wet'salted wt) Float ratio: Temperature: Enzyme concentration:

Lime concentration:

(gmsof enzyme preparation per .l0O-ml of lime liquor). The actual float ratio does not appear to be critical and can vary, for example, from l0Ol000%. Drum painting methods involving very small float ratios can be used. No critical float ratio has been found, but indications exist that the leather products obtained with the lowest float values may be slightly inferior. Similarly, wide varying lime concentrations from l-8% have been employed, depending on the properties'desired in the leather product.

The present enzymes are not limited to use'at the high pH levels of saturated lime, or lime with caustic soda or sodium carbonate. Theyare effectivealso at the lower pH values which correspond to buffered lime or other more acidic salts. ln passing it may be noted that other alkaline media such as the hydroxides and carbonates of sodium and potassium might be used in lieu of the lime to dehair, and these enzymes are usable therewith.

The temperature range for the liming-enzymatic dehairing may vary between about 15-40C. The nature of the enzymatic process favors the higher temperature, insofar as speed is concerned. lndications exist that the higher temperature operation seems to give more favorable results in terms of hair removal, but care should be taken in terms of the operating temperature to avoid temperature levels, eg in excess of 40C., at which the enzyme becomes inactivated rapidly, and at which damage to the hide may result.

The assistance of a -detergent, anionic or non-ionic in character has been found to help the unhairing. Suitable exemplary detergent quantities are l g per liter of float or about 0.05% wt of the wet salted skins or hides.

The enzyme proportions given above have been on a weight percent basis. However, the enzymes employed in the exemplary tests were only commercially pure products, far from a enzyme content. Their enzyme activity was approximately 6 KNPU/gm (KNPU is an abbreviation for 1000 NOVO-Proteolytic Units, a known standard. 1 KNPU is approximately equal to 0.3 Anson Units).

'Although the above examples describe tests'on cowhide, it may be noted that other hides and skins including for instance, calfskin, pigskin, sheepskin and goatskin can be treated with lime and these enzymes, using practices normal for lime alone. Thus dewooling of coarse wooled sheepskins which is not susceptible. to a highly alkaline pH bath method is commonlyaccomplished by a paint technique. Application of an enzyme containing paint to coarse wooled sheepskins is exemplified as follows:

Example VI Approximately 250 mls of a paint containing the following composition was applied to dried Australian sheepskins after an overnight soak in water:

2.5 g Sodium ch lorite 30 3 Enzyme l liter water l. A method for dehairing hides and skins which comprises treating the skins or hides with an aqueous alkaline medium having a pH above about pH-lO, said medium containing a proteolytic enzyme of the serine type showing optimal proteolytic activity against hemoglobin at a pH value above 9, and 80-100% of maximum activity at pH-l2, when measured by the Anson method, the said proteolytic enzyme being produced by aerobic cultivation of species of the genus Bacillus in a nutrient medium for production comprising assimilable carbon and nitrogen sources, using proteaseforming species of the said genus isolated from nature on a nutrient medium having a pH value within the range of 9 to 11, or mutants thereof, and maintaining the pH value of the said nutrient medium for production between 7.5 and 10.5 during the main part of the cultivation.

2. The method of claim 1 wherein the enzyme preparation content is from 0025-0. 10% by wt. of enzyme preparation based on the wet skins or hides.

3. The method of claim 1 wherein the medium has a pH in the range of about pH 1 l.0l3.0.

4. The method of claim 3 wherein the medium is a saturated solution of lime.

5. The method of claim 1 wherein the enzyme is recovered from a strain selected from 'NClB-8772, NCTC-4553, NClB-lOl47, NClB-l03 l 3 and NClB- 10317.

6. A method for dehairing hides and skins which comprises treating the skins or hides with an aqueous alkaline medium having a pH above about pH-lO 'said medium containing a proteolytic enzyme recovered from a strain of Bacillus alcalophilus.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. 1 3,840,433 DATED October 8, 1974 INVENTO I Knud Aunstrup et al It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 7, line 40 change "plexiglass" to read -PLEXIGLAS-.

Signed and Scaled this Thirrieth D ay of January I979 [SEA L| Arrest:

DONALD W. BANNER RUTH (T. MASON Arresting Oflicer Commissioner of Patents and Trademarks UNITED STAT S PATENT OFFICE CERTIFICATE OF CORRECTION 3,840,433 Dated October 8, 1974 Patent No.

Inventor( KNUD AUNSTRUP, OTTO ANDRESSEN & HELLE OUTTRUP It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

On the cover sheet, item number 73, change "Nono" to -Novo--.

Signed and sealed this 1st day of April 13375.

(SEAL) Attest:

, C. MARSHALL DANN RUTH C. MASON Commissioner of Patents Arresting Officer and Trademarks FORM PO-105O (10-69) uscoMM-Dc 60376-P69 Q n.5, GOVERNMENT PRINTING OFHCE II! 0-366-334

Referenced by
Citing PatentFiling datePublication dateApplicantTitle
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US5312749 *May 12, 1992May 17, 1994The United States Of America As Represented By The Secretary Of AgricultureIndustrial alkaline protease from shipworm bacterium
US5387518 *Apr 24, 1992Feb 7, 1995Showa Denko K.K.Alkaline protease having stability in solution with anionic surfactant, method for producing the same, use thereof and microorganism producing the same
US5474700 *Jan 14, 1994Dec 12, 1995The United States Of America As Represented By The Secretary Of AgricultureIndustrial alkaline protease from shipworm bacterium
US6375948 *Jul 11, 2000Apr 23, 2002Kao CorporationTreating method for suppressing hair growth
US6867032Dec 5, 2000Mar 15, 2005Daiwa Kasei K.K.Enzymatic unhairing agent for use in tanning for producing leather and method for enzymatic unhairing treatment
US7056499Feb 8, 2002Jun 6, 2006Kao CorporationFor suppressing the growth of the body hair, thereby decreasing the removing frequency of the body hair
US7211278Feb 13, 2004May 1, 2007Kao CorporationTreating method for suppressing hair growth
US7618801Oct 30, 2007Nov 17, 2009Danison US Inc.Streptomyces protease
US7879788Sep 29, 2009Feb 1, 2011Danisco Us Inc.Methods of cleaning using a streptomyces 1AG3 serine protease
US7985569May 31, 2007Jul 26, 2011Danisco Us Inc.Multiple substitutions, DNA encoding these proteases, vectors, host cells transformed with the vector DNA, and enzymes produced by the host cells; detergent compositions, animal feeds, textile and leather processing
US8455234Jun 16, 2011Jun 4, 2013Danisco Us Inc.Multiple mutation variants of serine protease
US8535927Nov 19, 2004Sep 17, 2013Danisco Us Inc.Micrococcineae serine protease polypeptides and compositions thereof
DE3429047A1 *Aug 7, 1984Feb 20, 1986Roehm GmbhEnzymatisches enthaarungsverfahren
EP1304389A1 *Dec 5, 2000Apr 23, 2003Daiwa Kasei Kabushiki KaishaEnzymatic unhairing agent for use in tanning for producing leather and method for enzymatic unhairing treatment
WO2008122640A2Apr 8, 2008Oct 16, 2008Novozymes AsAn enzymatic treatment of skin and hide degreasing
WO2009058679A1Oct 24, 2008May 7, 2009Danisco Us Inc Genencor DivStreptomyces protease
WO2011161135A1Jun 22, 2011Dec 29, 2011Novozymes A/SEnzyme dehairing of skins and hides
Classifications
U.S. Classification435/265, 435/221
International ClassificationC14C1/06, C14C1/00
Cooperative ClassificationC14C1/065
European ClassificationC14C1/06B