Can recombinant tree shrew (Tupaia belangeri chinensis) chymosin coagulate cow (Bos taurus) milk?

Genetically engineered chymosin from the tree shrew (Tupaia belangeri chinensis) has been obtained and partially characterized for the first time. The target enzyme was produced in Escherichia coli, strain BL21(DE3). It was shown that tree shrew recombinant chymosin coagulates cow milk (Bos taurus). The total and specific milk-clotting activity of the obtained enzyme was 0.7–5.3 IMCU/mL and 8.8–16.6 IMCU/mg. The nonspecific proteolytic activity of tree shrew recombinant chymosin in relation to total bovine casein was 30 and 117% higher than that of recombinant chymosin of cow and of single-humped camel respectively. It was found that in comparison with most of the known genetically engineered chymosins, the tree shrew enzyme showed exceptionally low thermal stability. After heating at 45°C, the coagulation ability of tree shrew recombinant chymosin decreased by more than 40%, and at 50°C the enzyme lost more than 90% of the initial milk-clotting activity. The Michaelis constant (Km), enzyme turnover number (kcat), and catalytic efficiency (kcat/Km) for genetically engineered chymosin from the tree shrew were 6.3 ± 0.1 µM, 11 927 ± 3169 s–1 and 1968 ± 620 µM–1 s–1, respectively. Comparative analysis showed that the primary structure of the chymosin-sensitive site of cow kappa-casein and the supposed similar sequence of tree shrew kappa-casein differed by 75%. The ability of tree shrew recombinant chymosin to coagulate cow’s milk, along with a low thermal stability and high catalytic efficiency with respect to the substrate, imitating the chymosin-sensitive site of cow kappa-casein, suggests that this enzyme is of potential interest for cheese making

Biochemical and technological properties of moose (<i>Alces alces</i>) recombinant chymosin

Recombinant chymosins (rСhns) of the cow and the camel are currently considered as standard milk coagulants for cheese-making. The search for a new type of milk-clotting enzymes that may exist in nature and can surpass the existing “cheese-making” standards is an urgent biotechnological task. Within this study, we for the first time constructed an expression vector allowing production of a recombinant analog of moose chymosin in the expression system of Escherichia coli (strain SHuffle express). We built a model of the spatial structure of moose chymosin and compared the topography of positive and negative surface charges with the correspondent structures of cow and camel chymosins. We found that the distribution of charges on the surface of moose chymosin has common features with that of cow and camel chymosins. However, the moose enzyme carries a unique positively charged patch, which is likely to affect its interaction with the substrate. Biochemical and technological properties of the moose rChn were studied. Commercial rСhns of cow and camel were used as comparison enzymes. In some technological parameters, the moose rChn proved to be superior to the reference enzymes. Сompared with the cow and camel rСhns, the moose chymosin specific activity is less dependent on the changes in CaCl2 concentration in the range of 1–5 mM and pH in the range of 6–7, which is an attractive technological property. The total proteolytic activity of the moose rСhn occupies an intermediate position between the rСhns of cow and camel. The combination of biochemical and technological properties of the moose rСhn argues for further study of this enzyme

МОБИЛЬНЫЕ ЭЛЕМЕНТЫ ГЕНОМА СОРТОВ TRITICUM. КОЛИЧЕСТВЕННЫЙ АНАЛИЗ С ПОМОЩЬЮ ПЦР «В РЕАЛЬНОМ ВРЕМЕНИ»

Исследовано относительное содержание ретротранспозонов Ty1/copia и 5.8S рДНК в геномах 9 сортов пшеницы. Разработана методика ПЦР с детекцией в режиме реального времени для выявления количества ретротранспозонов Ty1/copia и 5.8S рДНК. Выявлено повышенное относительное содержание ретротранспозонов в гексаплоидных сортах пшеницы по сравнению с тетраплоидными