Subtilin

It is to Jansen and Hirschmann (320) that we owe the discovery of an antibiotic product called subtilin in cultures (cells and media) of B. aubiilis. These workers pointed out several properties of the crude product which they isolated and later work showed that subtilin was a peptide. 

The extraction of subtilin from bacterial cells as well as from culture medium has been described by Dimick et al. (168,169), Lineweaver et al. (384), and Fevold et al. (195). In particular, the latter workers have investigated a method of extraction applicable to large quantities of submerged cultures. The culture is acidified to pH 2.0 by 3 N HCl, and then extracted with n-butanol. The subtilin is separated from the 

Crude preparations of subtilin can be fractionated by countercurrent distribution between water and n-butanol, or between water and secondary butanol (90). 

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Protected 3-methyl-D-cystein (257 Scheme 3.94), a structural unit of the peptide antibiotics nisin and subtilin, has been synthesized through the ring-opening of the aziridinecarbamide 254 with thiobenzoic acid (255) [143, 144]. The reaction took place overnight at room temperature and in methylene chloride to give 256 in greater than 95% yield. 

Section 5.08.4). Engineered expression systems have also been established for subtilin/ Pep5/" ° epidermin and gallidermin/ mutacin lacticin 3147 436,142 mersacidin. " " In these studies, the immunity genes were typically also required for generating successful expression systems. 

Klein, C. Entian, K.D. Cenes involved in self-protection against the lanti-biotic subtilin produced by Bacillus subtilis ATCC 6633. Appl. Environ. Microbiol., 60, 2793-2801 (1994)... 

A large number of a, 3-didehydro-a-amino acids have been identified as constituents of relatively low molecular weight cyclic compounds from microbial sources. However, the presence of a,p-didehydroalanine in bacterial as well as in mammalian histidine ammonia lyase and in phenylalanine ammonia lyase shows that the occurrence of a,p-didehydro-a-amino acids is not limited to small molecules alone 8 These residues are incorporated in natural sequences by posttranslation modification. a,p-Didehydro-a-amino acids have also been postulated to be precursors in the biosynthesis of several heterocyclic metabolites including penicillin and cephalosporin 9 Other well-known compounds containing ,( -di-dehydro-a-amino acids are nisin 10,11 (a food preservative112 ), subtilin (a broad spectrum antibiotic) 13 and some of the metabolites isolated from Streptomyces strains such as gri-seoviridin 14 ... 

From other bacteria nisin subtilin Streptococcus lactis P... 

Tolerance o/Lactococcus lactis to High Concentrations of Nisin Prepare cultures of Lactococcus lactis (ATCC 11454, NCIMB 8586) in sterile skim milk by incubating for 18 h at 30°. Prepare one or more flasks containing 100 mL of litmus milk, and sterilize at 121° for 15 min. Suspend 0.1 g of sample in the sterilized litmus milk, and allow to stand at room temperature for 2 h. Add 0.1 mL of the L. lactis culture, and incubate at 30° for 24 h. L. lactis will grow in this concentration of sample (about 1000 IU/mL) however, it will not grow in similar concentrations of other antimicrobial substances. This test will not differentiate Nisin from subtilin. 

Figure 14 Structures of nisins and subtilin. Dha, dehydroalanine Dhb, dehydrobutyrine Ala-S-Ala, lanthionine Abu-S-Ala, /3-methyllanthionine. Filled circles are substituted amino acids from the corresponding residues of nisin A.

Other bacteriocins are known, including subtilin [123]. This also inhibits outgrowth and enhances heat processes against some types of spores [124], Its mechanism of action and the basis for differing responses of spores, and of germinating and outgrowing spores, remain speculative. 

Tylosin is a macrolide produced by Streptomyces fradiae [125]. It is not sporicidal it increases the sensitivity of spores to heat and especially to ionizing radiation and inhibits outgrowth although at a later stage than nisin or subtilin [126],... 

Liu W, Hansen JN. The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus Cereus T spores and vegetative cells occurs by different mechanisms. Appl. Environ. Microbiol. 1993 59 648-651. 

The majority of these substances may be considered unique polypeptides which contain unusual amino acids or amino acids joined in some unusual manner. Antibiotics now definitely known to be in this class are the penicillins (2), gramicidin (2), tyrocidine (1), gramicidin-S 3), bacitracin (4), subtilin (-5), polymyxin (6), aerosporin (7) and others. The penicillins are produced by a mold. The others listed here are produced by related bacteria. Streptomycin is a very important antibiotic therapeutically but it does not belong to the polypeptide class, being a complex amino-sugar derivative. 

Subtilin is another polypeptide antibiotic produced by a different strain of B. suUilis. More than one antibiotic from this organism has been reported. We have not as yet studied this substance by counter-current distribution, but paper chromatography has indicated that it... 

Last but not least, it should be mentioned that a couple of peptidases have industrial importance. In particular, since subtilisins have a broad substrate specificity and are highly stable at neutral and alkaline pH they are of considerable industrial interest as protein-degrading additives to detergents. These reasons combined with their large data base make subtilisins attractive for protein engineering. Extensive engineering studies have been carried out on the Bacillus subtilins and more than 500 site-directed mutants have been produced to alter specific enzyme properties, such as pH profile, thermal stability or substrate specificity (see e. g. references[37 391). 

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