Process for the preparation of alkaline protease

Details Process for the preparation of alkaline protease Process for the preparation of alkaline protease

2002-03-13

2004-08-17

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S265000

Reexamination Certificate

active

06777219

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for the preparation of alkaline protease. More particularly, it relates to the preparation of the said alkaline protease using a fungal culture of the order entomophthorales. The present invention also relates to an eco-friendly application of the said protease in the pretanning processes of leather manufacture. Still more particularly, it relates to soaking, dehairing and bating of skins as well as dehairing of hides in leather manufacture.
BACKGROUND OF THE INVENTION
Proteases are the single class of enzymes, which occupy a pivotal position with respect to their physiological and commercial applications. Of the industrial enzymes, 75% are hydrolytic in nature. Proteases represent one of the three largest groups of industrial enzymes and account for 60% of the total worldwide sale of enzymes. Proteases have a variety of applications mainly in the detergent, food and pharmaceutical industries [Rao, M. B., Tanksale, M., Ghatage, M. S., and V. V. Deshpande, (1998) Microbiology and Molecular Biology Reviews. Vol.62, 597]. In view of the recent trend of developing environmentally friendly technologies, proteases are envisaged to have extensive applications in leather manufacture and in several bioremediation processes.
Leather processing involves several steps such as soaking, dehairing, bating and tanning. The major building blocks of skin and hair are proteinaceous matter. Conventional methods of leather processing employ chemicals such as sodium sulphide and lime which are highly polluting. Use of enzymes as alternatives to chemicals has proved successful in improving leather quality and in reducing environmental pollution [Puvanakrishanan, R. (1993) Science Reporter, 30 58].
Proteases are also used for selective hydrolysis of non-collagenous constituents of the skins and for removal of proteins such as albumins, globulins, elastins and reticulins.
Conventional method of dehairing consists of swelling of skins or hides under extremely alkaline conditions using lime followed by treatment with sulphide to solubilize proteins in significant reduction in both quantity of wastewater and in the pollution load.
Approximately 50% of the enzymes used as industrial process aids are proteolytic enzymes [Gorey. T., and Reichelt, J.(1983) industrial Enzymology: the application of enzymes in industry, Nature Press, N.Y]. Proteolytic and amylolytic enzymes derived from various sources viz. microbial, animal and plant sources have been applied individually or in combination to produce effective dehairing of hides and skins. Proteolytic enzymes are more efficient in enzymatic dehairing than amylolytic enzymes and hence find wider application.
Animal proteases like pancreatin have been utilized for the depilation of pigskins at a pH of 8.0-9.0 at 37° C. with pretreatment using sodium sulphate and sodium thiosulfate [Felicjaniak, B. (1975) Pr.Inst. Przem. Skorzanego, 19, 77]. Jonczyk and Studniarski (1985) have shown that pigskins are dehaired in 4.5 at 37° C. using a system containing a pancreatic enzyme preparation, ammonium sulphate and sodium bisulfate [Jonczyk, G. W. and Studniarski.K (1985) Przegl. Skorzany. 40, 222].
Plant proteases such as those obtained form powdered leaves and barks of the Jawasee shrub, which contains rich amounts of a proteolytic enzyme, are used for dehairing hides and skins in the Indian States of Gujarat, Rajasthan and Madhya Paradesh and a process for the manufacture of grain garment leather using Jawasee protease has been described [Yeshodha, K., Dhar, S. C. and Santappa, M. (1978) Leath. Sci., 25, 36].
A pineapple protease is found to have maximum activity at pH 3.5-4.5 and good hide dehairing effects are obtained at pH 5.0-5.5 in the temperature range of 30-42° C. [Xia Y. (1982) Pige Keji 5,8]. Adewoye and Bangaruswamy (1984) have developed a single unit process for dehairing of hides using the neutral protease from the fruit of
Adenopus breviflorus
[Adewoye, R. O. and Bangaruswamy, S, (1984) Leder, 35, 78].
With the renewed emphaisis on biotechnology, microbial enzymes have received increasing attention and processes that involve versatile utilization of microbial metabolic machinery for the production of enzymes are currently being studied with great interest. Unlike animal and plant proteases Microbial proteases can be produced in large quantities and genetic manipulation to increase activity is easier. As is well known, microbial proteases are derived from a wide variety of yeasts, molds and bacteria.
Several processes are reported for the production of fungal enzyme depilants and bates. The influence of several cultural conditions and important nutritional factors on the formation of protease by
A. parasiticus
on cheap indigenous materials has been shown [Bose, S. M. and Dhar, S. C. (1958) Indian Patent No. 64354]. Markin has recommended the use of enzyme preparations from
A. oryzae
and
A. flavus
for depilation [Markin, I. V (1963) Kozh Obuvn., Prom-st., 5, 30]. Recently, a fungal alkaline protease from
A. flavus
has been developed in Central Leather Research Institute, India and it has been found to be effective in the dehairing of skins [Malathi, S. and Dhar, S. C. (1991) Appl. Environ. Mircrobiol. 57, 712; Malathi, S., Chakraborty, R., Parthasarathy, K., Ramanaiah, B., Gupta, K. B and Mitra R. B., (1991) J. Amer. Leather Assoc, 86, 33].
Enzymes derived form bacteria have gained much commercial interest because of their easy production capabilities by submerged cultivation, high yield of enzyme, short duration of production and easy recovery of the enzyme. Proteolytic enzymes derived from a large number of Bacillus species have been reported to be used in dehairing and bating of hides and skins [Toyoda, H. and Futami, A. (1962) Bull. Japan Assoc. Leath. Technol., 8,49]. Detailed studies on the use of enzymes from
Bacillus subtilis
have been carried out by Simoncini and coworkers [Simoncini, A. Del Pezzo, L. Meduri, A. (1967) Cuoio Pelli Mat Concianti, 43, 382; Simoncini, A and Tessitore (1971), Cuoio Pello mat Cocianti 47, 201; Hameed, A., Walt, M. A. and Evens, C. S. (1996) World j. Microbiol. Biotechnol. 12, 289; Hameed, A., Walt, M. A. and Evans, C. S. (1996) J. Ind. Microbiol., 17, 77; Hameed, A., Keshavarz, T and Evans, C. S. (1999) J. Chem. Technol. Niotechnol. 74, 5].
A multiple protease concentrate from the culture filtrate of
Streptomyces moderatus
has been used for dehairing studies. While the individual proteases have been found to be ineffective in dehairing, the crude protein concentrate is efficient in the dehairing of hides and skins [Chandrasekaran, S. and Dhar, S. C. (1983) J. Ferment. Technol., 61, 511]. A novel method of depilation in an acid medium containing certain Lactobacillus culture has been reported [Schlosser, L. Kelller, W., Hein, A. and Heidemann, E., (1986) J. Soc. Leath. Technol. Chem. 70, 163]. The cattle hides or grain splits of raw hides are treated at 32° C. in culture medium obtained by inoculation with a stock culture of Lactobacillus in a plastic vessel. The culture is gently mixed for 1 min, once in an hour and 99% dehairing has been observed after incubation over one and half days.
Most commercial alkaline proteases used in detergents and leather applications are of bacterial origin viz. Novo-Nordisk, Denmark; M/s SPIC, Chennai, India & M/s Textan Chemicals, Chennai, India. Enzymes of fungal origin are advantageous due to the ease of cell removal during downstream processing. Although application of fungal proteases in leather processing has been reported it has not been studied on a commercial scale [Godfrey, T. and S. West (1996) Ind . Enzymology 296].
Production of alkaline protease using
Conidiobolus coronatus
has been reported by Tatsuaki and Koshi wherein the production of the protease was reported in complex medium containing glucose, organic and inorganic nitrogen sou

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