Obtaining of Recombinant Camel Chymosin and Testing Its Milk-Clotting Activity on Cow’s, Goat’s, Ewes’, Camel’s and Mare’s Milk

Abstract

In the cheese-making industry, commonly chymosin is used as the main milk-clotting enzyme. Bactrian camel (Camelus bactrianus) chymosin (BacChym) has a milk-clotting activity higher than that of calf chymosin for cow’s, goat’s, ewes’, mare’s and camel’s milk. A procedure for obtaining milk-clotting reagent based on recombinant camel chymosin is proposed here. Submerged fermentation by a recombinant yeast (Pichia pastoris GS115/pGAPZαA/ProchymCB) was implemented in a 50 L bioreactor, and the recombinant camel chymosin was prepared successfully. The activity of BacChym in yeast culture was 174.5 U/mL. The chymosin was concentrated 5.6-fold by cross-flow ultrafiltration and was purified by ion exchange chromatography. The activity of the purified BacChym was 4700 U/mL. By sublimation-drying with casein peptone, the BacChym powder was obtained with an activity of 36,000 U/g. By means of this chymosin, cheese was prepared from cow’s, goat’s, ewes’, camel’s and mare’s milk with a yield of 18%, 17.3%, 15.9%, 10.4% and 3%, respectively. Thus, the proposed procedure for obtaining a milk-clotting reagent based on BacChym via submerged fermentation by a recombinant yeast has some prospects for biotechnological applications. BacChym could be a prospective milk-clotting enzyme for different types of milk and their mixtures.

An impact of N-glycosylation on biochemical properties of a recombinant α-amylase from Bacillus licheniformis

“…The protein structure and enzyme activity are impacted by N-glycosylation. N-glycosylation of proteins produced in P. pastoris is noted, and this is followed by an increase in molecular weight [ 27 , 52 ]. In the present work, the effect of N-glycosylation on the α-amylase activity and thermal stability of α-amylase from B. licheniformis was determined, and the half-life at 80 °C is twofold shorter for glycosylated enzyme than for non-glycosylated analog.…”

Comprehensive bioinformatics-based annotation and functional characterization of bovine chymosin protein revealed novel biological insights

“…Recombinant technology has been used by researchers to engineer novel chymosin enzymes with tailored properties for specific applications ( Akishev et al, 2022 , Vallejo et al, 2012 , Wei et al, 2016 ). Notably, Vallejo et al demonstrated that recombinant goat chymosin displayed superior catalytic efficiency compared to buffalo, bovine, or camel versions ( Vallejo et al, 2012 ).…”

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

“…From 2001 to the present, the following rChns have been obtained and characterized: sheep (Ovis aries) [13], goat (Capra hircus) [14][15][16], buffalo (Bubalus arnee bubalis) [17,18], yak (Bos grunniens) [19,20], alpaca (Vicugna pacos) [21], Altai maral (Cervus elaphus sibiricus) [22][23][24][25], rabbit (Oryctolagus cuniculus) [26], white whale (Delphinapterus leucas) [27], two-humped camel (Camelus bactrianus) [28,29], and tupaya (Tupaia belangeri chinensis) [30].…”