# A Marine Alkaline Protease from Bacillus safensis DL12: Heterologous Expression, Purification and Preliminary Application in Animal Feed

## TL;DR

A marine enzyme from Bacillus safensis DL12 effectively reduces toxic gossypol in cottonseed meal, making it a promising tool for animal feed.

## Contribution

A novel marine alkaline protease is characterized and shown to efficiently detoxify cottonseed meal.

## Key findings

- The purified enzyme from B. safensis DL12 increased peptide content in cottonseed meal by 55.6%.
- The enzyme reduced free gossypol by 70% compared to the control, outperforming crude enzyme preparations.
- Optimal activity was observed at pH 8.0 and 50°C with enhancements from Fe2+, Cu2+, Ni2+, and DTT.

## Abstract

Cottonseed meal (CSM) is a cost-effective protein source, but its application is limited by the toxicity of free gossypol. Traditional physical and chemical detoxification methods are costly, energy-intensive, and cause nutrient loss, while microbial fermentation-based biological detoxification is considered more sustainable than chemical or physical approaches. This study reports an alkaline protease from the marine strain Bacillus safensis DL12 isolated from Yellow Sea sediments. Following cloning of its encoding gene and heterologous expression, enzymatic characterization of the purified enzyme revealed optimal activity at pH 8.0 and 50 °C, with Fe2+, Cu2+, Ni2+, and dithiothreitol (DTT) significantly enhancing its activity. Substrate hydrolysis analysis using the purified enzyme on soybean meal, peanut meal, rapeseed meal, and cottonseed meal demonstrated that, compared to the control group, cottonseed meal hydrolysates exhibited a 55.6% relative increase in peptide content and a 41.5% relative improvement in the degree of hydrolysis (DH), indicating higher hydrolysis efficiency among the four substrates. Notably, when hydrolyzing cottonseed meal with purified enzyme versus crude enzyme preparation at equivalent activity, the purified enzyme effectively reduced free gossypol content by 70% compared to the control, achieving more efficient detoxification than the crude enzyme preparation and most reported microbial treatments. These results highlight the potential of B. safensis DL12 protease as a marine-derived enzyme, offering promising prospects for enhancing protein digestibility and addressing the long-standing challenge of gossypol toxicity in cottonseed meal utilization.