Abstract
In this study, site-directed mutagenesis was carried out to alter properties of Mucor pusillus rennet (MPR) in order to find a potential substitution of commercial chymosin. Mutant G186D/E13D screened from thousands of mutants showed a significant milk-clotting activity (MCA). Mutant G186D/E13D rennet was purified and characterized. The molecular weight was estimated to be 44 kDa by SDS-PAGE. The maximum enzyme activity was at a wide range of pH (5.0-7.0) and 60ºC. The enzyme was inhibited by metal ions (Fe2+, Fe3+, Cu+ and Zn2+), 1.10-Phenantrolin and pepstatin A. Further texture analysis of types of cheddar cheese made by non-mutant rennet, mutant (G186D/E13D) rennet and commercial rennet suggested that the soluble nitrogen content and hardness of cheddar cheese made by chimeric mutant rennet was decreased without any significant change in flavor between these cheeses. The result implicated that, to some extent, the mutant rennet could decrease hydrolysis of protein during ripening of cheese, probably as a candidate for a useful milk coagulant.
Highlights
Chymosin is an aspartic proteinase (EC 3.4.23.4) that is responsible for the coagulation of milk in the fourth stomach of unweaned calves in the form of an inactive precursor prochymosin [1], which is used extensively in cheese production because it cleaves κ-casein in a specific manner, at the Phe105-Met106 bond, with low proteolytic activity, and for the production of quality cheeses with good flavor and texture [2]
The proteolysis of cheeses was assayed by determination of total nitrogen (TN), pH 4.6 phosphotungstic acid-soluble nitrogen (PTASN) according to methods described by Christensen et al [36]
Site 186 and 13 of Mucor pusillus rennet (MPR) are key positions, which relate to temperature sensitivity and substrate specificity
Summary
Chymosin is an aspartic proteinase (EC 3.4.23.4) that is responsible for the coagulation of milk in the fourth stomach (abomasum) of unweaned calves in the form of an inactive precursor prochymosin [1], which is used extensively in cheese production because it cleaves κ-casein in a specific manner, at the Phe105-Met106 bond, with low proteolytic activity, and for the production of quality cheeses with good flavor and texture [2]. Unavailability of calf stomach and ethical problems associated with animal slaughtering has necessitated the finding of other alternatives to calf chymosin In this regard, various plants and microbial proteases alternatives are used for chymosin production. Plant sources for milk-clotting enzymes have been identified from Cynara scolymus [3], Carica papay [4], Streblus asper [5], Centaurea calcitrapa [6] and Albizia [7] Most of these sources are not suitable for production of quality cheese as they produce a bitter taste [2]. We have cloned the preproRMPP gene, and have developed efficient expression systems for the enzymes as zymogens in Pichia pastoris [24] By using this system, site-directed mutagenesis of many milk-clotting enzymes was carried out to generate mutant enzymes with amino acid exchanges at position 13, 101 and 186. Replacement of Glu13/Gly186 was found to cause a marked decrease in the proteolytic activity
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