以副溶血弧菌生物膜基质为靶点，采用96孔板结晶紫染色法筛选出纤维素酶、脂肪酶、蛋白酶k为单酶，将单酶组合为复合酶，在单因素实验的基础上，采用响应面分析法优化组合酶浓度。通过结晶紫染色、菌落计数、扫描电镜等，评价组合酶对304不锈钢上副溶血弧菌生物膜的抑制作用。响应面优化得到的组合酶中纤维素酶浓度为1.00 mg/mL，脂肪酶浓度为1.10 mg/mL，蛋白酶k浓度为1.50 μg/mL时生物膜抑制率最大，为89.73%，比优化之前的各单酶相比，对副溶血弧菌生物膜的抑制率有显著性提高，与预测基本相符，说明模型拟合良好，组合酶中各单酶浓度准确可行。优化得到的组合酶有效抑制了304不锈钢上副溶血弧菌生物膜的生长，抑制率为59.35%，扫描电镜图也证实了复合酶的抑制效果。生物膜降解酶复合有效提升了对生物膜的抑制率，为致病菌生物膜的降解及消除提供了新的思路。

Vibrio parahaemolyticus biofilm matrix was used as the target, and cellulase, lipase and protease K were screened and selected as single enzymes by the crystal violet staining assay using 96-well plates. These single enzymes were combined to form composite enzymes. On the basis of single factor experiments, response surface methodology was used to optimize the concentration of the combined enzymes. The inhibitory actions of the combined enzymes against Vibrio parahaemolyticus biofilm on 304 stainless steel were evaluated by the crystal violet staining assay, colony counting and scanning electron microscopy. The composite enzymes obtained by response surface optimization with 1.00 mg/mL of cellulase, 1.10 mg/mL of lipase and 1.50 μg/mL of protease K exhibited the highest inhibition rate (89.73%). The rate of inhibition for the composite enzyme against the Vibrio parahaemolyticus biofilm significantly increased compared with the single enzyme, which was generally in accordance with the predicted value. These results indicated that the model fitted well and the concentration of each enzyme in the composite enzyme was accurate and feasible. The optimized composite enzymes inhibited effectively the growth of Vibrio parahaemolyticus biofilm on 304 stainless steel with the inhibition rate as 59.35%. Examinations by scanning electron microscopy also confirmed the inhibitory effect of the composite enzymes. The composite enzymes can effectively increase the rate of inhibition against biofilm, which provides new ideas for the degradation and elimination of pathogenic bacterial biofilms.

山东省重点研发计划项目(2019GNC106064)