CIESC Journal ›› 2012, Vol. 63 ›› Issue (3): 887-893.DOI: 10.3969/j.issn.0438-1157.2012.03.030

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Screening and identification of phospholipase-producing strains and their applications

JIANG Fangyan1, WANG Jinmei2, DAI Dazhang1, LI Chun1,2   

  • Received:2011-07-06 Online:2012-03-05 Published:2012-03-05

产磷脂酶菌株的筛选鉴定及其应用

姜芳燕1,王金梅2,戴大章1,李春1,2   

  1. 1北京理工大学生命学院;2石河子大学化学化工学院
  • 通讯作者: 李春

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

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