本研究利用新型交联剂京尼平制备了枯草杆菌碱性蛋白酶交联聚集体（BAP-CLEAs）。以酶活回收率为指标，确定了BAP-CLEAs制备的最佳条件为：交联剂质量浓度0.50%，交联温度35 ℃，交联时间12 h，此时BAP-CLEAs的酶活回收率为55.04%。采用扫描电镜及红外光谱对BAP-CLEAs进行表征，结果证明枯草杆菌碱性蛋白酶在京尼平的作用下成功交联。与游离酶相比，BAP-CLEAs的最适pH值向碱性方向偏移，由9.4变为10.3，在较宽的pH范围和温度范围内保持较高的酶活。另外，在2%浓度的酪蛋白底物中重复使用5次后，BAP-CLEAs还能保持86.42%的酶活性。以上催化特性的结果表明，枯草杆菌碱性蛋白酶在京尼平的作用下可成功交联形成酶聚集体，且该交联酶聚集体具有比游离酶更优越的pH稳定性、温度稳定性和重复使用稳定性，有良好的工业应用前景。

In this study, Genipin as a novel crosslinking agent was used to prepare the crosslinked Bacillus subtilis alkaline protease aggregates (BAP-CLEAs). Using the recovery rate of enzyme activity as the main indicator, the optimal processing conditions for the preparation of BAP-CLEAs were determined: mass concentration of the crosslinking agent 0.50%, crosslinking temperature 35 ℃ and crosslinking time 12 h. The recovery rate of the enzyme activity of the BAP-CLEAs prepared under the optimum condition was 55.04%. The characterization results of the BAP-CLEAs by SEM and FTIR showed that Bacillus subtilis alkaline protease was successfully crosslinked with the aid of genipin. Compared with the free enzyme, the optimal pH of BAP-CLEAs shifted towards the alkaline direction (from 9.4 to 10.3), and a relatively high enzyme activity was maintained in wide pH and temperature ranges. In addition, BAP-CLEAs still had 86.42% of the initial enzyme activity after 5 repeated uses in a casein substrate at 2%. The above catalytic characteristics indicated that the Bacillus subtilis alkaline protease could be successfully crosslinked to form CLEAs with the aid of genipin. The BAP-CLEAs had superior pH stability, temperature stability and repeatability compared with the free enzyme, thus has a good industrial application prospect.

广东省应用型科技研发专项资金项目（2015B020230001）；国家重点研发计划项目专项（2016YFC0400702-5）；广东省重点领域研发计划项目（2019B020222001）