Novel FRET-substrates of Rhizomucor pusillus rennin: Activity and mechanistic studies.

The development of sensitive, easy and reliable methods for the determination of Rhizomucor pusillus rennin (MPR) activity, in free and in immobilized form, along with the elucidation of the mechanism of action, represent challenges for the widespread use of the enzyme in industrial cheese production. These could be accomplished by using highly specific and sensitive substrates, as well as direct assay methods. We designed and synthesized novel substrates based on Fluorescence Resonance Energy Transfer (FRET) for the MPR by employing computational simulation techniques and peptide synthesis in liquid phase. Three FRET-substrates (Abz-GFY-pNA, Abz-SFY-pNA and Abz-GFI-pNA) were found active, while the Abz-GFY-pNA showed the highest reliability, sensitivity and specificity among them. Subsequently, a novel mechanism of MPR action was elucidated, with the development of novel methods for assaying activity in free and immobilized form, which both may contribute in the wider use of rennin in cheese production and other biotechnological applications.

Immobilized rennin in TC/SG composite in cheese production.

The object of the current study was to develop a new process for continuous Feta-type cheese production using a biocatalyst consisting of immobilized rennin on a tubular cellulose/starch gel (TC/SG) composite, which has been proven to be an appropriate carrier for enzyme immobilization. Different methodologies were used in order to prepare four biocatalysts. The most effective was selected for cheese production in a 1L continuous system, providing two economically useful results for the dairy industries: (i) increase of productivity by the continuous coagulation of milk, and (ii) saving of the rennin enzyme expenses of the batch coagulation of milk. The criteria used to choose the appropriate biocatalyst was based on the time of coagulation in successive batches, the concentration of immobilized rennin combined with the filter efficiency and its application in the continuous system. Physicochemical analyses of the cheeses at various stages of the ripening were performed. No significant differences compared to cheeses prepared with the traditional method were found. Aroma compounds were determined by SPME GC-MS.

Tubular cellulose/starch gel composite as food enzyme storehouse.

The objective of this study was to produce a composite biocatalyst, based on porous cellulosic material, produced after wood sawdust delignification (tubular cellulose; TC) and starch gel (SG), for the development of bioprocesses related to enzyme applications. The composite biocatalyst was studied by Scanning Electron Microscopy to observe the SG deposition in the TC pores, and porosimetry analysis to determine the average pore diameter and surface area. The deposition of SG into the TC tubes provided a TC/SG composite with reduced pore sizes. X-ray powder diffractometry showed a decrease of crystallinity with increased SG ratio in the composite. The composite was used as an insoluble carrier for entrapment of the dairy enzyme rennin, leading to the production of an active biocatalyst for milk coagulation (initiation of milk clotting at about 20 min and full coagulation at about 200 min), creating perspectives for several applications in food enzyme research and technology.