Abstract
Chymosin (also known as rennin) plays an essential role in the coagulation of milk in the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is of high cost. Here, we present an alternative method to producing bovine chymosin from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from bovine, was introduced into the tobacco nuclear genome under control of the viral 35S cauliflower mosaic promoter. The integration and transcription of the foreign gene were confirmed with Southern blotting and reverse transcription PCR (RT-PCR) analyses, respectively. Immunoblotting analyses were performed to demonstrate expression of chymosin, and the expression level was quantified by enzyme-linked immunosorbent assay (ELISA). The results indicated recombinant bovine chymosin was successfully expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total soluble protein. The tobacco-derived chymosin exhibited similar native milk coagulation bioactivity as the commercial product extracted from bovine rumen.
Highlights
Chymosin, more commonly known as rennin, is a key industrial enzyme used to produce cheese, a historically important food in Western countries and one that is in increasing demand in Eastern countries such as China
Cheeses are distinguished by their flavors and/or textures which depend both on their composition and the chymosin used in cheese processing to coagulate the milk [1,2,3,4]
Van Rooijen et al [35] claimed that the optimized bovine prochymosin gene is suitable to expression in at least 22 plant species listed in the patent, but they only showed the details in flax and oilseed rape
Summary
More commonly known as rennin, is a key industrial enzyme used to produce cheese, a historically important food in Western countries and one that is in increasing demand in Eastern countries such as China. AA qquuaannttiittaattiivvee aannaallyyssiiss bbyy EELLIISSAA iinnddiiccaatteedd tthhaatt eexxpprreessssiioonn lleevveellss vvaarriieedd ffrroomm 1188..11ttoo8833..55nngg//gg ffrreesshh wweeiigghhtt,, mmeeaanniinngg 00..1188%% ttoo 00..5522%% ooff TTSSPP ((FFiigguurree 33BB)). Van Rooijen et al [35] claimed that the optimized bovine prochymosin gene is suitable to expression in at least 22 plant species listed in the patent, but they only showed the details in flax and oilseed rape This helped to increase the percentage of chymosin in the total seed protein of flax (up to around 2.6% TPS) and oilseed rape (average of 4.43% TPS). The chymosin extracted from tobacco leaves exhibited milk-clotting activity, which implies that the enzyme had been activated in the plant tissue, as the pH value of crude extract and milk are approximately 7.0 and 6.5 respectively, which is outside the range of 4.0 to 5.0 for self-activation, providing indirect evidence that the recombinant chymosin has been correctly transited to the intercellular space . The presence of active chymosin added to the significance of this work: The crude extract could be directly applied to milk for cheese processing without protein purification, and this might reduce the production cost
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