Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin

Abstract

Moose (Alces alces) recombinant chymosin with a milk-clotting activity of 86 AU/mL was synthesized in the Kluyveromyces lactis expression system. After precipitation with ammonium sulfate and chromatographic purification, a sample of genetically engineered moose chymosin with a specific milk-clotting activity of 15,768 AU/mg was obtained, which was used for extensive biochemical characterization of the enzyme. The threshold of the thermal stability of moose chymosin was 55 °C; its complete inactivation occurred after heating at 60 °C. The total proteolytic activity of moose chymosin was 0.332 A₂₈₀ units. The ratio of milk-clotting and total proteolytic activities of the enzyme was 0.8. The K_m, k_cat and k_cat/K_m values of moose chymosin were 4.7 μM, 98.7 s^-1, and 21.1 μM^-1 s^-1, respectively. The pattern of change in the coagulation activity as a function of pH and Ca²⁺ concentration was consistent with the requirements for milk coagulants for cheese making. The optimum temperature of the enzyme was 50-55 °C. The introduction of Mg²⁺, Zn²⁺, Co²⁺, Ba²⁺, Fe²⁺, Mn²⁺, Ca²⁺, and Cu²⁺ into milk activated the coagulation ability of moose chymosin, while Ni ions on the contrary inhibited its activity. Using previously published data, we compared the biochemical properties of recombinant moose chymosin produced in bacterial (Escherichia coli) and yeast (K. lactis) producers.

Keywords: Michaelis–Menten kinetics; calcium chloride concentration; coagulation specificity; milk-clotting activity; moose; proteolytic activity; recombinant chymosin; substrate pH; temperature optimum; thermal stability.

Figure 1

Genetic map of the pSVB-Alc plasmid vector: ICL—regulatory sequence of isocitrate lyase promoter; GAP—core sequence of glyceraldehyde-3-phosphate dehydrogenase promoter; α-factor secretion signal—α-MCE S. cerevisiae protein secretion signal; Alc—moose prochymosin sequence; CYC—CYC1 S. cerevisiae transcription terminator sequence; ADH1—alcohol dehydrogenase gene promoter; ZeoR—zeocin resistance gene; ICL-5′—5′ isocitrate lyase promoter gene sequence; ori—the replication origin; AmpR—ampicillin resistance gene.

Figure 2

Productivity of producer strain (Alc-D K. lactis) in terms of moose rProChn under various cultivation conditions: (a) dependence of milk-clotting activity on glucose concentration: 1—series 1 (1% glucose); 2—series 2 (2% glucose); 3—series 3 (3% glucose); (b) typical dependence of MA on the duration of cultivation at the initial glucose concentration of 3%.

Figure 3

Milk-clotting activity of recombinant chymosins (rChns) from different animal sources (1—rChn-Bos; 2—rChn-Alc-KL; 3—rChn-Cam) at different temperatures (30–65 °C).

Figure 4

Total proteolytic activity (A₂₈₀) of recombinant chymosins (rChn) (1—rChn-Cam; 2—rChn-Bos; 3—rChn-Alc-KL) after different incubation periods (0–180 min) of enzyme–substrate mixtures.

Figure 5

Proteolytic specificity of recombinant chymosins: 1—molecular weight markers; 2—control 1 (substrate without heating); 3—milk + rChn-Bos; 4—milk + rChn-Cam; 5—milk + rChn-Alc-KL; 6—control 2 (substrate heated at 35 °C for 60 min). On the left, molecular weights are indicated in kDa. On the right, the bands of α-, β-, κ-CN, and para-κ-CN (MM ≈ 16 kDa, tracks 3–5) are indicated. An asterisk (*) shows polypeptide components with MM << 14 kDa (track 5).

Figure 6

Milk-clotting activity (MA, %) of recombinant chymosins (rChns) (1—rChn-Bos; 2—rChn-Cam; 3—rChn-Alc-KL) at various substrate temperatures (25–65 °C).

Figure 7

Milk-clotting activity (in terms of duration of coagulation, %) of recombinant chymosins (rChns) (1—rChn-Alc-KL; 2—rChn-Cam; 3—rChn-Bos) at various pH (6.0–7.0) of the milk substrate.

Figure 8

Milk-clotting activity (in terms of duration of coagulation, %) of recombinant chymosins (rChns) (1—rChn-Bos; 2—rChn-Cam; 3—rChn-Alc-KL) at various concentrations of calcium chloride (0.0–5.0 mM) in milk.