Protease alkaline

In 1958, the microbial alkaline protease Alcalase (Novo Industries) was produced by fermentation of a strain of Bacillus licheniformis. It had high StabiHty and activity at pH 8—10, was marketed in 1961, and was incorporated into Bio 40. However, it was not until the successful marketing of the presoaking agent Biotex in 1963 that detergent manufacturers saw the tme possibiHties of enzymes. 

Several new detergent enzymes have emerged on the market (Table 1). Truly alkaline proteases, introduced in 1974 and 1982, were fermented on strains of Bacillus lentus firmus. These enzymes have a pH optimum between 9 and 11, and have taken important market shares from Alcalase. 

In 1989, two enzymes based on genetic engineering techniques were introduced, ie, a cloned alkaline protease (IBIS) and a protein engineered Subtihsin Novo (Genencor, California). Lipase and ceUulase types of detergent enzymes have also begun to appear. 

Fig. 15. En2ymatic hydrolysis of wheat gluten at 72.5°C and pH 7.5 by an alkaline protease from Bacillus licheniformis. The numbers on the curves are en2yme—substrate ratios (E/S) in activity units (AU)/kg of protein where S = 7.4% (N x 5.7).

Boumann, U., et al. Three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa, a two-domain protein with a calcium binding parallel beta roll motif. EMBO J. 12 3357-3364, 1993. 

Alkaline protease from Bacillus subtilis DY, pH 8, 37°, 80-85% yield. Methyl esters are cleaved similarly. 

Certain proteases, for example alkaline protease from Bacillus suhtilis, have also proven to be good catalysts for the Michael addition of nitrogen nucleophiles to a,/3-efhylenic compounds [118, 119]. 

There has been increasing interest in these on account of their potential for the synthesis of valuable enzymes that include alkaline proteases and those that degrade starch and cellulose. Strains of Bacillus sp. have been most extensively examined (Horikoshi 1999). 

Protein isolation with affinity precipitation has been discussed in detail by Mattiasson and co-workers (see, e.g. Galaev and Mattiassion, 1997) and they have provided an exhaustive tabulation. Polymers varied from alginate.s/chitosan to dextran to NIPAM. Examples of the used proteins are from trypsin, p-glucosidase, xylanase, alkaline protease, etc. It is remarkable that affinity precipitation can sometimes give results comparable to affinity chromatography. 

Recovery of alkaline protease from fermentation broth [82]... 

Thus the alkaline protease obtained from Bacillus licheniformis with a molecular mass of about 27 000 consists of 274 amino acid residues and has serine and histidine as active sites. Pancreatic trypsin with a molecular mass of about 24 000 contains 230 amino acid residues and also has serine and histidine as active sites. Papain (molecular mass about 23 000 and 211 amino acid residues) has cysteine and histidine as active sites. 

The first structures of this kind were reported in 1993 pectate lyase G from Erwinia chrysanthemi (Yoder et al, 1993) and alkaline protease from Pseudomonas aeruginosa (Baumann et al, 1993). Based on consideration of these crystal structures, the term parallel //-helix was introduced for a fold containing three //-strands per coil, and parallel //-roll for a fold with two //-strands per coil (Baumann etal, 1993 Yoder andjurnak, 1995 Yoder et al., 1993). The epithet parallel was intended to emphasize the distinction between these folds and the previously observed helical structure of the antibiotic gramicidin which contains both l- and D-amino acids and... 

H 0 A-butanoyl-L-homoserine lactone, BHL or C4-HSL Aeromomas hydrophila Aeromonas salmonicida Pseudomonas aeruginosa, Serratia liquefaciens Extracellular protease, biofilm formation. Extracellular protease. Virulence factors - alkaline protease, cyanide, elastase, haemolysin, lectins, pyocyanin, rhaminolipid, RpoS Swarming, protease. 

A r-(3-oxododccanoyl)-L-homoscrinc lactone, OdDHL or 30, C12-HSL Pseudomonas aeruginosa Virulence factors - alkaline protease, clatasc, exotoxin A, haemolysin, neuraminidase, exoenzyme S, Xcp secretion, RhlR, biofilm formation. 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

Dehairing is carried out using alkaline proteases such as subtilisin in a very alkaline bath. Alkaline conditions tend to swell the hair roots, so easing the removal of the hair by allowing the proteases to selectively attack protein in the hair follicle. Other specific enzymes are used for skins from particular species. 

In order to prove enzyme engineering feasibility, it was important to develop a model system. One of the prime considerations for any model would be the commercial potential of the model. Table I lists the major commercial enzymes and the market size in US dollars (5). The alkaline proteases (subtilisins) are clearly the major single class of enzymes in commercial use today, representing 25% of the total enzyme market of 600 million. The primary use of subtilisins is as additives in laundry detergents to aid in the removal of proteinaceous stains from cloth. 

Various genes in aflatoxin biosyndre sis cluster Alkaline protease gene... 

Moser, M., Menz, G., Blaser, K., and Crameri, R. (1994). Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus. Infect. Immun. 62, 936-942. 

Griffin, H.L. Greene, R. V. Cotta, M. A. (1992) Industrial alkaline protease fran shipworm bacteria. US Pat. Appl. US 880,912, 15 Sqrt. to US Dqrt Agriculture. Chan. Abstr., 1993,118, 142526e. 

Mukherjee, A. K., Adhikari, H., Rai, S. K. (2008). Rroduction of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrical grass and potato peel as low-cost medium Characterization and application of enzyme in detergent formulation. Biochem. Engg. J.,39, 353-361. 

Katano, S., Oki, T., Matsuo, Y., Yoshihira, K., Nara, Y., Miki, T., Matsui, T., and Matsumoto, K. (2003). Antihypertensive effect of alkaline protease hydrolysate of the pearl oyster Pinctada fucata martencii separation and identification of angiotensin-I converting enzyme inhibitory peptides. Nippon Suisan Gakk. 69, 975-980. 

Matsufuji, H., Matsui, T., Seki, E., Osajima, K., Nakashima, M., and Osajima, Y. (1994). Angiotensin I-converting enzyme inhibitory peptides in an alkaline protease hydrolyzate derived from sardine muscle. Biosci. Biotechnol. Biochem. 58, 2244-2245. 

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