Abstract: Using soybean phospholipids as substrates, one strain which expressed phospholipase was screened out from the oil-rich soil sample by preliminary screening of plate cultivation and re-screening of shake flask fermentation. According to the morphological characteristics, physiochemical properties, and 16S rRNA sequences, the strain BIT-18 was identified as Pseudomonas fluorescens. Synthetic phosphatecholine (1-palmitoyl-2-oleoyl phosphatecholine) was used as substrate, then the fatty acid compositions of hydrolyzates were analyzed by gas chromatography, and the phospholipase from BIT-18 was determined as phospholipase B. This enzyme could not tolerate a high temperature. The optimal conditions for the enzymatic reaction were 25℃ and pH 6. 5, and low concentrations of metal ions were good for trigging its reaction. The phospholipase B from BIT-18 was used as catalyst for the degumming of soybean oil in a self-made batch reactor. The optimal conditions were phospholipase B dosage of 500 U·kg^-1 and 2% water, and reaction at 40℃, pH 4.7 for 6 h. The residual phosphorus content of degummed oil decreased from 90.1 mg·kg^-1 to 4.6 mg·kg^-1, with the degumming rate as high as 94.9%, which showed a good application potential in the degumming of vegetable oils.Using soybean phospholipids as substrates, one strain which expressed phospholipase was screened out from the oil-rich soil sample by preliminary screening of plate cultivation and re-screening of shake flask fermentation. According to the morphological characteristics, physiochemical properties, and 16S rRNA sequences, the strain BIT-18 was identified as Pseudomonas fluorescens. Synthetic phosphatecholine (1-palmitoyl-2-oleoyl phosphatecholine) was used as substrate, then the fatty acid compositions of hydrolyzates were analyzed by gas chromatography, and the phospholipase from BIT-18 was determined as phospholipase B. This enzyme could not tolerate a high temperature. The optimal conditions for the enzymatic reaction were 25℃ and pH 6. 5, and low concentrations of metal ions were good for trigging its reaction. The phospholipase B from BIT-18 was used as catalyst for the degumming of soybean oil in a self-made batch reactor. The optimal conditions were phospholipase B dosage of 500 U·kg^-1 and 2% water, and reaction at 40℃, pH 4.7 for 6 h. The residual phosphorus content of degummed oil decreased from 90.1 mg·kg^-1 to 4.6 mg·kg^-1, with the degumming rate as high as 94.9%, which showed a good application potential in the degumming of vegetable oils.

Key words: phospholipase B, screening, identification, enzymatic degumming;Pseudomonas fluorescensfont-family: "Times New Roman", mso-bidi-font-size: 12.0pt, mso-bidi-font-family: 宋体, mso-font-kerning: 1.0pt, mso-ansi-language: EN-US, mso-fareast-language: ZH-CN, mso-bidi-language: AR-SA, mso-fareast-font-family: 宋体, mso-hansi-font-family: 宋体">phospholipase B, screening, identification, enzymatic degumming;Pseudomonas fluorescens

摘要： 采用磷脂平板初筛和摇瓶复筛，从富油土样中筛选得到一株产磷脂酶菌株BIT-18。经菌株形态特征、生理生化特征及16S rRNA序列分析，鉴定其为荧光假单胞菌（Pseudomonas fluorescens）。以磷脂标准品（1-棕榈酰-2-油酰-Sn-甘油-3-磷脂酰胆碱）为底物，通过气相色谱分析反应产物的脂肪酸成分，定性鉴定P.fluorescens BIT-18表达的磷脂酶为B型磷脂酶。该酶为低温酶，最适温度和pH值分别为25℃和6.5，低浓度的金属离子有利于其酶促反应进行。以磷脂酶B为催化剂在自制间歇式反应器中对大豆油进行酶法脱胶，在加酶量500 U·kg^-1，加水量2%，温度40℃，pH 4.7的条件下反应6 h，脱胶油磷含量由90.1 mg·kg^-1降至4.6 mg·kg^-1，脱胶率高达94.9%，显示出良好的应用前景。

关键词: 磷脂酶B, 筛选, 鉴定, 酶法脱胶, 荧光假单胞菌