YS performed the Y. pestis studies. KN-L and KDR participated in the design of the study. EH-G conceived of the study, participated

in its design and coordination, and helped draft the manuscript. All AMPK inhibitor authors read and approved the final manuscript.”
“Background Rennet, a milk coagulant from the abomasum of milk-fed calves and lambs, is the industrial gold standard in cheese manufacturing [1]. Chymosin is the major milk-clotting component of natural rennet preparations obtained from young animals as its activity amounts to 90% of the total observed potency [2]. However, due to increased demand in cheese products, ARN-509 in vitro animal-derived milk coagulants are not sufficient to cover the production. Therefore, the demand has prompted increased research efforts in the manufacture of recombinant and microbial rennin [3]. Nevertheless, the rennin of microbial origin might be contaminated by other enzymes which might affect cheese ripening by causing bitterness during storage

[4]. Until now, several aspartic proteinases (APs) such as pepsin, rennin, renin, and cathepsin D have been extensively studied. Microbial APs such as rhizopuspepsin and penicillopepsin have been reported to be either intracellular or extracellular enzymes with most of them having been cloned and purified. For example, acid proteinase from Metschnikowia reukaufii[5] has been cloned and expressed in Escherichia coli while three clt genes encoding milk-clotting proteinases from Myxococcus xanthus have been cloned and expressed in E. coli, Saccharomyces cerevisiae and P. pastoris[3]. It is also known that CRT0066101 molecular weight fungal extracellular thermophilic proteinases from R. miehei and R. pusillus

are still used as substitutes for calf chymosin in cheese manufacturing [6]. However, the enzymes are extensively proteolytic which may result in impaired cheese organoleptic characteristics. Moreover, the proteinases are resistant to heat treatment compared to bovine chymosin and thus can remain active in the curd for longer periods of time [7]. Additionally, proteinases with low heat stability have been observed in M. varians[8] and M. circinelloides[9]. Although studies on the characterization of an acid proteinase from M. circinelloides were performed, the molecular characteristics of the enzyme remain unknown. Resveratrol The mentioned thermo-labile proteinases may provide technological advantages for industrial utilization. In this work, the cloning and expression of the aspartic proteinase from M. circinelloides was performed. Methods Fungal strain, bacterial strains and plasmids The microorganism used as the source of the gene encoding MCAP was M. circinelloides strain DSM 2183 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). E. coli strain TOP10 [10] was used to amplify the plasmids carrying the cloned gene. E.