DEHAIRING OF LEATHER
United States Patent 3840433
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.
US Patent References:
Production of leather
Wallerstein et al. - May 1936 - 2041731
PREPARATION OF PROTEOLYTIC ENZYMES HAVING MAXIMUM ACTIVITY AT HIGH ALKALINITY
Aunstrup et al. - July 1962 - 3674643
/3679548.html
Donovan et al. - July 1972 - 3679548
PROTEOLYTIC ENZYMES, THEIR PRODUCTION AND USE
Aunstrup et al. - March 1973 - 3723250
Production of leather
Wallerstein et al. - May 1936 - 2041731
PREPARATION OF PROTEOLYTIC ENZYMES HAVING MAXIMUM ACTIVITY AT HIGH ALKALINITY
Aunstrup et al. - July 1962 - 3674643
/3679548.html
Donovan et al. - July 1972 - 3679548
PROTEOLYTIC ENZYMES, THEIR PRODUCTION AND USE
Aunstrup et al. - March 1973 - 3723250
Inventors:
Aunstrup, Knud (Farum, DK)
Andressen, Otto (Copenhagen, DK)
Outtrup, Helle (Varlose, DK)
Andressen, Otto (Copenhagen, DK)
Outtrup, Helle (Varlose, DK)
Application Number:
05/251646
Publication Date:
10/08/1974
Filing Date:
05/09/1972
View Patent Images:
Export Citation:
Assignee:
Nono, Terapeutisk Laboratorium A/s (Bagsvaerd, DK)
Primary Class:
Other Classes:
435/221
International Classes:
C14C1/06; C14C1/00; C14C1/06; C12B1/00
Field of Search:
195/6,65,62,66R
Primary Examiner:
Shapiro, Lionel M.
Attorney, Agent or Firm:
Fidelman, Wolffe, Leitner & Hiney
Parent Case Data:
This application is a continuation-in-part of prior applications Ser. No. 761,546 filed Sept. 23, 1968 now Pat. No. 3,723,250 and Ser. No. 772,830 filed Nov. 1, 1968 now Pat. No. 3,674,643.
Claims:
What is claimed is
1. 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-10, 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-12, 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 protease-forming 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 0.025-0.10% 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 11.0-13.0.
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 NCIB-8772, NCTC-4553, NCIB-10147, NCIB-10313 and NCIB-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-10, said medium containing a proteolytic enzyme recovered from a strain of Bacillus alcalophilus.
1. 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-10, 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-12, 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 protease-forming 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 0.025-0.10% 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 11.0-13.0.
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 NCIB-8772, NCTC-4553, NCIB-10147, NCIB-10313 and NCIB-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-10, said medium containing a proteolytic enzyme recovered from a strain of Bacillus alcalophilus.
Description:
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 10-13.0 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 removal of 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 by-product, 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. 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 a pH 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 11, 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 80-100% of maximum activity when measured at pH 12 by the Anson method. A readily identified group of proteolytic enzymes which may be incorporated are the proteolytic enzymes obtained by the cultivation of 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 inventor's 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 11, 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 has, also been made of a number of different enrichment methods.
Enrichment methods are known in the art. Reference can be made to Hayaishi, Methods in Enzymology, Vol. 1, 126-131. One principle is to let a sample from nature grow on a nutrient medium having a specific and selected composition favoring the growth of a microorganism giving metabolic products having the properties aimed at. Another principle is to store the sample from nature together with a compound, such as an inorganic salt, favoring the development of the desired microorganism, cfr. M. A. El-Nakeeb and H. A. Lechevalier, Appl. Microbiol, Vol. 11, 75 (1963), and thereafter to spread the sample on a suitable nutrient medium adjusted on a pH-value within the range of 8 to 12.
Subsequent cultivated of the microorgansims with 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 (1959). One Anson unit means throughout this specification the amount of proteolytic enzyme digesting hemoglobin at pH-value of 10.1 and a temperature of 25°C during a reaction time of 10 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 milli-equivalent 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-range referred to, and recovering from the medium the proteolytic enzymes formed during the cultivation.
Experiments seem to show that it is convenient to carry out the cultivation at a pH-value of the culture medium within the range 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 Sept. 23, 1968. The enzymes of particular interest for deharing purposes are those showing 80-100% of maximum activity when measured at pH 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.
Another preferred embodiment of the invention is use of the proteolytic enzymes available from Bacillus alcalophilus.
For exemplary purposes, this species and the proteolytic enzymes therefrom will be described below in detail.
In the Dutch periodical "Antonie van Leeuwenhoek", 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 2 CO 3 , KHCO 3 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 Bergey's 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 Edinburgh under the NCIB number 8772, where the strain by a mistake had been considered as a Bacillus subtilis.
During their metabolism, the strains of the species Bacillus alcalophilus produce novel proteolytic enzymes extracellularly, that such enzymes may be recovered from the cultivation media and that they show a pronounced activity at high alkalinities, which makes 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 11, 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 1-5%, 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 the cultivation. 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.
Besides the nutrient medium should contain the usual trace substances.
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 40°C. is convenient. The temperature is preferably about 30 to 37°C.
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 NCIB 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 25°C. during a reaction time of 10 minutes with such an initial velocity that per minute there 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 tap water Saccharose 50 g do. Barley flour 50 g do. Soybean meal 20 g do. Sodium caseinate 10 g do. Na 2 HPO 4 12H 2 O 9 g do. 2) Medium BSX with the following composition: Barley flour 100 g per liter of tap water Soybean meal 30 g do.
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 for 90 minutes at 120°C., and after the autoclaving the pH value was adjusted to 9.3-10.3 by addition of sodium carbonate or sodium sesquicarbonate in a concentration of 0.2 M and 0.1 M, respectively. There were used four flasks for each bacterium, and samples from the culture media for determining enzyme content expressed in Anson units were taken after cultivation in 3, 4, 5 and 6 days respectively. The flasks were, during the cultivation, placed on a rotating table with 220 revolutions per minute, and the temperature during the cultivation was 30°C.
The maximum proteolytic activity measured in Anson units per kg of the nutrient medium appears in Table 1 below.
Table I ______________________________________ Bacillus Maximum proteolytic Final pH alcalophi- Nutrient activity value lus medium Anson units per kg ______________________________________ NCIB 8772 BPFA 14 7.7 25 7.7 BSX 6 8.8 2.5 8.2 NCTC 4553 BPFA 16 7.9 13 7.6 ______________________________________
By fractionated precipitation with ethyl alcohol enzyme preparations in powder form were prepared as it appears from the following Table II.
Table II ______________________________________ NCIB NCTC Bacillus alcalophilus 8772 4553 ______________________________________ Starting kg of culture broth 0.10 0.25 material Anson units per kg 7.6 12 Anson units in total 0.76 3 1st preci- g Kieselguhr 3 0.75 pitation ml C 2 H 5 OH 150 385 2nd preci- g Kieselguhr 2.5 3 pitation ml C 2 H 5 OH 370 770 g powder 4.4 5.3 Anson units per g Yield (measured at pH 10.1) 0.14 0.3 Anson units in total 0.62 1.6 % 81 53 ______________________________________
The strain NCIB 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 30°C., the velocity of the agitator 570 rpm and the aeration 0.25 m 3 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 10.1.
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, 10, 11 and 12 at 25°C., and expressed in percentage of maximum activity. The results are compiled in Table III below.
Table III ______________________________________ pH value ______________________________________ 7 7.5 8 9 10 11 12 ______________________________________ 50 75 85 100 NCIB 8772 33 54 67 71 85 100 44 70 80 100 NCTC 4553 34 52 64 72 79 100 ______________________________________
The activity was also measured at a constant pH value of 10.1 and varing temperatures, viz. 25, 37, 50, 60, 66 and 75° of Celsius. The results of these measurements are compiled in Table IV below.
Table IV ______________________________________ Temperature °C. ______________________________________ 25 37 50 60 66 75 ______________________________________ NCTC 4553 % activity at pH 10.1 14 32 65 100 26 2 ______________________________________
In general, the enzyme preparations used for practice of the invention consist of a solid or liquid mixture of the proteolytic enzymes and other components; they can consist of granules into which the enzymes are incorporated; for instance, together with other enzymes or substances having other than enzymatic activity useful for the utility of the enzyme compositions. When the enzymes are not used in crystalline form, they may be accompanied by impurities of organic nature, such as proteins and carbohydrates from the culture medium. The enzyme powders may contain stabilizers. The enzyme compositions in liquid form can constitute solution or suspensions which may contain stabilizers, if necessary.
The alkali stable enzyme preparations or compositions for dehairing use normally show an enzyme content not exceeding about 10% by weight. In some cases, however, the enzyme content may be considerably higher. The following experiments made with preferred enzymes are intended to illustrate the utility of the alkaline stable enzymes for dehairing purposes under high pH.
A salted cowhide (the butt) is sliced in pieces measuring about 20 × 4 cm. The pieces are steeped 24 hours and fat and meat are scraped off. The pieces of hide are then placed in 400 ml of different enzyme solutions contained in glasses having a volume of 500 ml. The glasses are incubated at 30°C. for 24 hours. The pieces are then removed from the solutions and the hairs are scraped off with a piece of plexiglass. The dehairing effect is evaluated in accordance with the following scale:
1. Easy and complete removal of the hairs
2. Easy removal of the hairs, but spots of hairs remained on the hide
3. No or difficult removal of the hairs
The proteolytic enzyme solutions used contain 1 g calcium hydroxide per 130 g of water. The amount of enzymes appears from the below Table V, also indicating the pH values at the beginning and the end of the dehairing process together with the results thereof.
Table V ____________________________________________________________ ______________ No. 1 2 3 4 ____________________________________________________________ ______________ Enzyme from control C 303 C 367 C 372 strain NCIB 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 11.8 11.8 11.8 11.9 11.8 Dehairing result 3 1 1 2 2 3 1 ____________________________________________________________ ______________
The enzyme C-303 (NCIB 10147) was tested against a standard detergent proteolytic enzyme i.e. Alcalase, for initial activity at 25°C. at various pH levels, and for residual activity after 24 hours at various pH levels. The substrate was denatured hemoglobin. The results may be seen in the hereto attached drawing wherein:
FIG. 1 shows the effect of pH on activity; and
FIG. 2 shows the pH stability of the same two enzymes.
As may be seen in FIG. 1 the pH optimum for the preferred dehairing enzyme is 9.5 but the enzyme is almost equally active up to pH 12. The activity of the reference enzyme drops off rapidly with pH to an apparent 60% residual activity at pH 12, but this level of activity is transitory. As may be seen in FIG. 2, essentially all the activity of the reference enzyme has been destroyed by 24 hours at pH in excess of about pH 11.5. The enzyme C-303 (NCIB-10147) still retains approximately 60% of its initial activity after 24 hours at pH 12. The pH stability curves of FIG. 2 demonstrate the incompatibility of the prior art proteolytic enzymes with the liming procedure. Although the pH stability tests were not conducted under dehairing circumstances, the pH test results are consistent with dehairing experience.
To repeat, the leather art is committed to liming. A combination liming and enzyme treatment can be carried out effectively with the alkali stable enzymes.
For further understanding of the present invention, the following specific examples thereof are presented. All employ the enzyme C-303.
Example I
Cowhides soaked according to conventional procedures were drummed for 3 hours, then for 5 minutes each hour for 24 hours under the following conditions:
Float ratio: 200% Temperature: 25-27°C. Enzyme concentration: 0.2%(wt.% per volume of float) Lime concentration: 4% (on wet salted hide wt)
The hair was completely loose in 24 hours. After subsequent processing, the finished leather was identical in physical properties to a set of lime-sulfide treatment controls but had slightly greater area yield.
Example II
Cowhides were drummed intermittently for 24 hours under the following conditions:
Float ratio: 250% Temperature: 23-25°C. Enzyme concentration: 0.1% (wt% per volume of float) Lime concentration: 2% (on wet salted hide wt) Sodium sulfite con.: 1% (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 III
Cowhides were drummed intermittently for 24 hours using the following conditions, after which period the hair was loose.
______________________________________ Float ratio: 150% Temperature: 20°C. Enzyme concentration: 0.075% (wt% per volume of float) Lime concentration: 4% (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: 600% Temperature: 30°C. Enzyme concentration: 0.1% (wt% per volume of float) pH: 10.0
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.
Example V
54 kgs of hide were treated according to a drum painting technique under the following conditions:
Float ratio: 20% Temperature: 28°C. Enzyme concentration: 0.1% (on the wet salted wt) Lime concentration: 4% (on the wet salted wt)
After 6 or 18 hours the float was made up to 200% and after a further 2 hours intermittent drumming, the hair was completely loose.
Overall the tests described in Examples I-V demonstrate the extent to which a combined lime-enzymatic dehairing is feasible. The enzyme preparation concentration may vary relatively widely, from 0.025-1.0 wt% of the wet salted hides or skins. Thus the enzyme content in the lime liquor may be from about 0.05-0.4% (gms of enzyme preparation per 100 ml of lime liquor). The actual float ratio does not appear to be critical and can vary, for example, from 100-1000%. 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 1-8% 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. They are effective also at the lower pH values which correspond to buffered lime or other more acidic salts. In 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 15-40°C. The nature of the enzymatic process favors the higher temperature, insofar as speed is concerned. Indications 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, e.g. in excess of 40°C., 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 1 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 100% 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 commonly accomplished 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:
30 g Enzyme 2.5 g Sodium chlorite 400 g Ca(OH) 2 , 1 liter water
The skins were painted, then folded once and stored at 30°C. After 24 hours the skins could be easily pulled. They were then vegetable tanned; the leather was found quite satisfactory.
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 removal of 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 by-product, 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. 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 a pH 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 11, 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 80-100% of maximum activity when measured at pH 12 by the Anson method. A readily identified group of proteolytic enzymes which may be incorporated are the proteolytic enzymes obtained by the cultivation of 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 inventor's 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 11, 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 has, also been made of a number of different enrichment methods.
Enrichment methods are known in the art. Reference can be made to Hayaishi, Methods in Enzymology, Vol. 1, 126-131. One principle is to let a sample from nature grow on a nutrient medium having a specific and selected composition favoring the growth of a microorganism giving metabolic products having the properties aimed at. Another principle is to store the sample from nature together with a compound, such as an inorganic salt, favoring the development of the desired microorganism, cfr. M. A. El-Nakeeb and H. A. Lechevalier, Appl. Microbiol, Vol. 11, 75 (1963), and thereafter to spread the sample on a suitable nutrient medium adjusted on a pH-value within the range of 8 to 12.
Subsequent cultivated of the microorgansims with 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 (1959). One Anson unit means throughout this specification the amount of proteolytic enzyme digesting hemoglobin at pH-value of 10.1 and a temperature of 25°C during a reaction time of 10 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 milli-equivalent 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-range referred to, and recovering from the medium the proteolytic enzymes formed during the cultivation.
Experiments seem to show that it is convenient to carry out the cultivation at a pH-value of the culture medium within the range 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 Sept. 23, 1968. The enzymes of particular interest for deharing purposes are those showing 80-100% of maximum activity when measured at pH 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.
Another preferred embodiment of the invention is use of the proteolytic enzymes available from Bacillus alcalophilus.
For exemplary purposes, this species and the proteolytic enzymes therefrom will be described below in detail.
In the Dutch periodical "Antonie van Leeuwenhoek", 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 2 CO 3 , KHCO 3 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 Bergey's 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 Edinburgh under the NCIB number 8772, where the strain by a mistake had been considered as a Bacillus subtilis.
During their metabolism, the strains of the species Bacillus alcalophilus produce novel proteolytic enzymes extracellularly, that such enzymes may be recovered from the cultivation media and that they show a pronounced activity at high alkalinities, which makes 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 11, 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 1-5%, 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 the cultivation. 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.
Besides the nutrient medium should contain the usual trace substances.
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 40°C. is convenient. The temperature is preferably about 30 to 37°C.
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 NCIB 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 25°C. during a reaction time of 10 minutes with such an initial velocity that per minute there 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 tap water Saccharose 50 g do. Barley flour 50 g do. Soybean meal 20 g do. Sodium caseinate 10 g do. Na 2 HPO 4 12H 2 O 9 g do. 2) Medium BSX with the following composition: Barley flour 100 g per liter of tap water Soybean meal 30 g do.
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 for 90 minutes at 120°C., and after the autoclaving the pH value was adjusted to 9.3-10.3 by addition of sodium carbonate or sodium sesquicarbonate in a concentration of 0.2 M and 0.1 M, respectively. There were used four flasks for each bacterium, and samples from the culture media for determining enzyme content expressed in Anson units were taken after cultivation in 3, 4, 5 and 6 days respectively. The flasks were, during the cultivation, placed on a rotating table with 220 revolutions per minute, and the temperature during the cultivation was 30°C.
The maximum proteolytic activity measured in Anson units per kg of the nutrient medium appears in Table 1 below.
Table I ______________________________________ Bacillus Maximum proteolytic Final pH alcalophi- Nutrient activity value lus medium Anson units per kg ______________________________________ NCIB 8772 BPFA 14 7.7 25 7.7 BSX 6 8.8 2.5 8.2 NCTC 4553 BPFA 16 7.9 13 7.6 ______________________________________
By fractionated precipitation with ethyl alcohol enzyme preparations in powder form were prepared as it appears from the following Table II.
Table II ______________________________________ NCIB NCTC Bacillus alcalophilus 8772 4553 ______________________________________ Starting kg of culture broth 0.10 0.25 material Anson units per kg 7.6 12 Anson units in total 0.76 3 1st preci- g Kieselguhr 3 0.75 pitation ml C 2 H 5 OH 150 385 2nd preci- g Kieselguhr 2.5 3 pitation ml C 2 H 5 OH 370 770 g powder 4.4 5.3 Anson units per g Yield (measured at pH 10.1) 0.14 0.3 Anson units in total 0.62 1.6 % 81 53 ______________________________________
The strain NCIB 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 30°C., the velocity of the agitator 570 rpm and the aeration 0.25 m 3 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 10.1.
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, 10, 11 and 12 at 25°C., and expressed in percentage of maximum activity. The results are compiled in Table III below.
Table III ______________________________________ pH value ______________________________________ 7 7.5 8 9 10 11 12 ______________________________________ 50 75 85 100 NCIB 8772 33 54 67 71 85 100 44 70 80 100 NCTC 4553 34 52 64 72 79 100 ______________________________________
The activity was also measured at a constant pH value of 10.1 and varing temperatures, viz. 25, 37, 50, 60, 66 and 75° of Celsius. The results of these measurements are compiled in Table IV below.
Table IV ______________________________________ Temperature °C. ______________________________________ 25 37 50 60 66 75 ______________________________________ NCTC 4553 % activity at pH 10.1 14 32 65 100 26 2 ______________________________________
In general, the enzyme preparations used for practice of the invention consist of a solid or liquid mixture of the proteolytic enzymes and other components; they can consist of granules into which the enzymes are incorporated; for instance, together with other enzymes or substances having other than enzymatic activity useful for the utility of the enzyme compositions. When the enzymes are not used in crystalline form, they may be accompanied by impurities of organic nature, such as proteins and carbohydrates from the culture medium. The enzyme powders may contain stabilizers. The enzyme compositions in liquid form can constitute solution or suspensions which may contain stabilizers, if necessary.
The alkali stable enzyme preparations or compositions for dehairing use normally show an enzyme content not exceeding about 10% by weight. In some cases, however, the enzyme content may be considerably higher. The following experiments made with preferred enzymes are intended to illustrate the utility of the alkaline stable enzymes for dehairing purposes under high pH.
A salted cowhide (the butt) is sliced in pieces measuring about 20 × 4 cm. The pieces are steeped 24 hours and fat and meat are scraped off. The pieces of hide are then placed in 400 ml of different enzyme solutions contained in glasses having a volume of 500 ml. The glasses are incubated at 30°C. for 24 hours. The pieces are then removed from the solutions and the hairs are scraped off with a piece of plexiglass. The dehairing effect is evaluated in accordance with the following scale:
1. Easy and complete removal of the hairs
2. Easy removal of the hairs, but spots of hairs remained on the hide
3. No or difficult removal of the hairs
The proteolytic enzyme solutions used contain 1 g calcium hydroxide per 130 g of water. The amount of enzymes appears from the below Table V, also indicating the pH values at the beginning and the end of the dehairing process together with the results thereof.
Table V ____________________________________________________________ ______________ No. 1 2 3 4 ____________________________________________________________ ______________ Enzyme from control C 303 C 367 C 372 strain NCIB 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 11.8 11.8 11.8 11.9 11.8 Dehairing result 3 1 1 2 2 3 1 ____________________________________________________________ ______________
The enzyme C-303 (NCIB 10147) was tested against a standard detergent proteolytic enzyme i.e. Alcalase, for initial activity at 25°C. at various pH levels, and for residual activity after 24 hours at various pH levels. The substrate was denatured hemoglobin. The results may be seen in the hereto attached drawing wherein:
FIG. 1 shows the effect of pH on activity; and
FIG. 2 shows the pH stability of the same two enzymes.
As may be seen in FIG. 1 the pH optimum for the preferred dehairing enzyme is 9.5 but the enzyme is almost equally active up to pH 12. The activity of the reference enzyme drops off rapidly with pH to an apparent 60% residual activity at pH 12, but this level of activity is transitory. As may be seen in FIG. 2, essentially all the activity of the reference enzyme has been destroyed by 24 hours at pH in excess of about pH 11.5. The enzyme C-303 (NCIB-10147) still retains approximately 60% of its initial activity after 24 hours at pH 12. The pH stability curves of FIG. 2 demonstrate the incompatibility of the prior art proteolytic enzymes with the liming procedure. Although the pH stability tests were not conducted under dehairing circumstances, the pH test results are consistent with dehairing experience.
To repeat, the leather art is committed to liming. A combination liming and enzyme treatment can be carried out effectively with the alkali stable enzymes.
For further understanding of the present invention, the following specific examples thereof are presented. All employ the enzyme C-303.
Example I
Cowhides soaked according to conventional procedures were drummed for 3 hours, then for 5 minutes each hour for 24 hours under the following conditions:
Float ratio: 200% Temperature: 25-27°C. Enzyme concentration: 0.2%(wt.% per volume of float) Lime concentration: 4% (on wet salted hide wt)
The hair was completely loose in 24 hours. After subsequent processing, the finished leather was identical in physical properties to a set of lime-sulfide treatment controls but had slightly greater area yield.
Example II
Cowhides were drummed intermittently for 24 hours under the following conditions:
Float ratio: 250% Temperature: 23-25°C. Enzyme concentration: 0.1% (wt% per volume of float) Lime concentration: 2% (on wet salted hide wt) Sodium sulfite con.: 1% (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 III
Cowhides were drummed intermittently for 24 hours using the following conditions, after which period the hair was loose.
______________________________________ Float ratio: 150% Temperature: 20°C. Enzyme concentration: 0.075% (wt% per volume of float) Lime concentration: 4% (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: 600% Temperature: 30°C. Enzyme concentration: 0.1% (wt% per volume of float) pH: 10.0
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.
Example V
54 kgs of hide were treated according to a drum painting technique under the following conditions:
Float ratio: 20% Temperature: 28°C. Enzyme concentration: 0.1% (on the wet salted wt) Lime concentration: 4% (on the wet salted wt)
After 6 or 18 hours the float was made up to 200% and after a further 2 hours intermittent drumming, the hair was completely loose.
Overall the tests described in Examples I-V demonstrate the extent to which a combined lime-enzymatic dehairing is feasible. The enzyme preparation concentration may vary relatively widely, from 0.025-1.0 wt% of the wet salted hides or skins. Thus the enzyme content in the lime liquor may be from about 0.05-0.4% (gms of enzyme preparation per 100 ml of lime liquor). The actual float ratio does not appear to be critical and can vary, for example, from 100-1000%. 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 1-8% 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. They are effective also at the lower pH values which correspond to buffered lime or other more acidic salts. In 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 15-40°C. The nature of the enzymatic process favors the higher temperature, insofar as speed is concerned. Indications 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, e.g. in excess of 40°C., 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 1 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 100% 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 commonly accomplished 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:
30 g Enzyme 2.5 g Sodium chlorite 400 g Ca(OH) 2 , 1 liter water
The skins were painted, then folded once and stored at 30°C. After 24 hours the skins could be easily pulled. They were then vegetable tanned; the leather was found quite satisfactory.