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Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the Lactococcus lactis extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of Lactococcus lactis as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of ␣ S1 -casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.Proteolysis and its secondary reactions play a major role in the maturation of Cheddar and many other bacterium-ripened cheese varieties (16). Proteolysis in Cheddar cheese is a complex process that involves endogenous milk enzymes, coagulant, and microbial proteinases and peptidases. Hydrolysis of intact casein (CN) is catalyzed almost exclusively by the added coagulant and endogenous milk enzymes, while proteinases and peptidases from Lactococcus lactis starter bacteria and adventitious (nonstarter) lactic acid bacteria are responsible for the production of water-soluble peptides and free amino acids (17). The contribution of individual enzymes in the cheese matrix to this process will also be influenced by specificity, relative activity, stability in the cheese matrix, and in the case of intracellular enzymes, access to appropriate substrates.In many bacterium-ripened cheeses, the L. lactis cell envelope-associated proteinase (lactocepin, EC 3.4.21.96) is the most important microbial enzyme for the conversion of largemolecular-weight (water-insoluble) peptides produced by coagulant or plasmin into the small water-soluble peptides needed for flavor development (10,17,35). Lactocepin is a 180-to 190-kDa membrane-anchored enzyme that belongs to the subtilisin family of serine proteases. Although lactocepins exhibit more than 98% amino acid sequence identity, purified enzymes may be differentiated by their relative affinity and specificity for individual CNs (23). Genetic studies showed that most differences in lactocepin specificity could be traced to amino acid substitutions in the enzyme substrate-binding regions, and this ...

Murine monoclonal antibodies reactive with the major sigma subunit (sigma-70) of Escherichia coli RNA polymerase were obtained by standard hybridoma techniques. Western blot analyses established that seven antibodies had unique specificities after various chemical and enzymatic methods were used to fragment sigma. Peptides were purified by HPLC using size-exclusion, reverse-phase, or ion-exchange chromatography. The epitopes for six of these antibodies have been localized to specific peptides. These peptides were further characterized by amino acid composition and N-terminal sequencing. Sigma, which has a molecular weight of 70.2K, runs as 83K on SDS gels in this study. This anomalous behavior has been localized to the very acidic N-terminal half of the molecule. One antibody is unable to bind to native sigma. Two others do not bind well to sigma when it is contained in holoenzyme, indicating that their epitopes are in regions of sigma which are inaccessible in the holoenzyme complex. All three of these antibodies fail to inhibit in vitro transcription by holoenzyme. The other four antibodies all can inhibit in vitro transcription.

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The primary structure of sperm histone H1parecrJinus has been determined. H1par,c~Jinus consists of a polypeptide chain of the following 248 amino acid residues : Pro-Gly-Ser-Pro-Gln-Lys-Arg-Ala-AlaSer-Pro-Arg-Lys-Ser-Pro-Arg-Lys-Ser-Pro-Lys-Lys-Ser-Pro-Arg-Lys-Ala-Ser-Ala-Ser-Pro-Ala-Lys-Ala-Ala-Ala-Lys-Arg -Lys-Ala-Ala-Leu-Ala-LysLys-Lys -Ala-Ala-Ala-Ala-Lys-Arg-Lys-Ala-Ala -Ala-Lys -Ala-Lys -Lys-Ala-Lys-Lys -Pro -LysLys-Lys-Ala-Ala-Lys-Lys-Ala-Lys-Lys-Pro-Ala-Lys-Lys-Ser-Pro-Lys-Lys-Ala-Lys-Lys-ProAla-Lys-Lys-Ser-Pro-Lys-Lys-Lys-Lys-Ala-Lys-Arg-Ser-Pro-Lys-Lys-Ala-Lys-Lys-Ala-Ala-Ser-Pro-Lys-Lys-Ala-Arg-Lys. The protein consists of three domains. Compared to other HI and H5 histones, there is a very similar hydrophobic central domain and the carboxyl-terminal domain is very rich in lysine and alanine. Hlparectrcnus is similar to H5 histones in that the carboxyl-terminal domain also contains many arginine residues close to the carboxyl terminus. The carboxyl-terminal domain of HI P~~~~F~,~~ appears to have been constructed by a series of variable duplications. The amino-terminal domain of H~P~~~~J ,~~~~ is longer and quite different to that of other HI and H5 histones and is characterized by a repeating tetrapeptide of the general type Ser-Pro-(basic)z.The known sequence of a histone H1 gene from Psammechinus miliaris [Schaffner, W. et al. (1978) Cell, 14, 655-6711 is compared to the sequence of H1parecfJinus. Again the central hydrophobic domains are similar whereas the amino terminal domains are very different. Arg LysThe functions of the various domains of sperm histone H1parec~linus are discussed.In the preceding paper [l] the sequence of the first 84 residues of the sea urchin histone H1 have been reported. That investigation included a set of four cyanogen bromide fragments generated from the N-terminal region of the protein. The fifth CNBr peptide, CN-I, comprises 169 residues out of a total of 248 in the protein. The composition of CN-I is unusual insofar as 49 residues are alanine and 75 lysine and arginine. Such a composition indicates the presence of rather monotonous sequences, consisting possibly of an arrangement of reiterations of alanine lysine-rich stretches.Only few handles for breaking up this large cyanogen bromide fragment became evident from the composition of the fragment. The elucidation of the primary structure of this 169-residue fragment to be