# Comparative biochemical properties of recombinant goat and calf chymosins and their implications in dairy processing

## TL;DR

This study compares the effectiveness of recombinant goat and calf chymosins in clotting goat and cow milk for cheese production.

## Contribution

The first comparison of recombinant goat and calf chymosin biochemical properties in goat milk.

## Key findings

- Recombinant goat chymosin showed maximum activity at 60°C in both goat and cow milk.
- Goat chymosin had 13.6% higher activity in goat milk compared to cow milk.
- Both enzymes efficiently clot sheep, cow, and camel milk but poorly clot mare milk.

## Abstract

The nutritional and functional properties of goat milk are well-established. However, the use of camel and calf-derived chymosins during cheese making from goat milk results in relatively low productivity. This study aimed to produce recombinant goat chymosin and investigate its biochemical properties and milk-clotting activity in goat and cow milk. To achieve this, recombinant goat chymosin was successfully expressed in Pichia pastoris, yielding 148,000 units/L of enzyme activity. Recombinant goat chymosin has exhibited maximum enzyme activity at 60 °C in both milk types, whereas recombinant calf chymosin showed maximum activity at 65 °C for goat milk and 75 °C for cow milk. Moreover, goat chymosin exhibited 13.6% higher activity in goat milk than that in cow milk. Both the enzymes efficiently clot sheep, cow, and camel milk but exhibited low activity against mare milk. The milk-clotting activity of both the enzymes was remarkably influenced by the calcium chloride concentration. To our knowledge, this study is the first to compare the biochemical properties of recombinant goat and calf chymosin in goat milk. The high specific activity of recombinant goat chymosin in goat milk suggests its potential as an effective milk-clotting enzyme in goat milk cheese production.

The online version contains supplementary material available at 10.1038/s41598-025-11833-x.