Aim:  The study of a milk‐clotting protease secreted by Bacillus licheniformis strain USC13. Methods and Results:  Growth of B. licheniformis USC13 in LB medium resulted in the production of a serine protease with a molecular weight of 62 kDa processed to its mature form of 34 kDa, both forms were found in the extracellular medium. The enzyme exhibited typical milk‐clotting kinetics. Conclusions:  The capacity of this protease to produce milk curds could make it useful as a new source of milk coagulants. Significance and Impact of the Study:  Cheese‐making industry seeks for novel enzyme sources, and microbial coagulants have several advantages over animal and plant counterparts. The protease from B. licheniformis has the ability to produce milk curds although more studies about quality of both the enzyme and the milk curds formed should be carried out in the future to confirm its usefulness in the dairy industry.

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There is no analytical method currently available in Spain to determine elapsed time in wine aging. The only control used is based on the levels of esters and other secondary metabolites as important indicators related to wine quality. The main goal of this work was to establish a useful method to differentiate among young, "crianza", "reserva" and "gran reserva" wines based on the study of the enzymatic decay of different hydrolases present in the wines and also to determine which enzyme(s) may be applicable in fraud detection as regards time and storage conditions of the wine. We elaborated red wine based on "Tempranillo" grapes from the Rioja Alta Alavesa and fermented with Saccharomyces cerevisiae USC 013, followed by malolactic fermentation by Oenococcus oeni CECT 630 in order to evaluate the different enzymes. One of the enzymes present, i.e., invertase, which is resistant to inactivation, could be a useful marker of wine aging.

A new allele, the EPG1-2 gene, which codes for an endopolygalacturonase in Kluyveromyces marxianus CECT1043, has been cloned. The gene has 1086 bp and the protein 362 amino acids, one more than the previously described Epg1p. Epg1-2p shows a high degree of similarity with the polygalacturonases of fungi and yeasts. The sequences common to all of the polygalacturonases of prokaryotes, fungi, and higher plants are also conserved in Epg1-2p. The EPG1-2 gene has been expressed in Pichia pastoris , and, when fused with the signal peptide of the alpha-factor of Saccharomyces cerevisiae , the protein is properly secreted into the media. The recombinant enzyme does not appear to be fully glycosylated by P. pastoris or not glycosylated in the same manner as in K. marxianus, but it maintains the same optimum temperature (55 degrees C) and pH (4.5) and the same stability at different temperatures and pH values as the native enzyme, also showing the same hydrolytic behavior. The recombinant strain produces 200-fold more enzyme than the wild-type strain of K. marxianus, making it a yeast of potential industrial interest for the production of endopolygalacturonase for the food industry.