Bacterial strain capable of producing phosphatidase C, and screening method thereof
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A screening method and phospholipid production technology, applied in the field of microorganisms, can solve the problems of deviation of true activity, low enzyme production, interference with nitrophenol determination, etc., and achieve the effect of convenient operation
Active Publication Date: 2014-01-15
JIANGNAN UNIV
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Problems solved by technology
Therefore, using PPC as a natural phospholipid analog as an activity evaluation standard has a certain deviation from its true activity.
[0006] In addition, the enzyme production of wild strains is generally low, and the fermentation broth often has a certain color, which will also interfere with the determination of p-nitrophenol when NPPC is the substrate.
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preparation example Construction
[0026] Preparation of primary screening medium:
[0027] Egg yolk LB agar medium: Take 10ml of 50% egg yolk liquid and add it to 90ml of LB agar medium that has been sterilized and cooled to about 55°C, shake well while it is hot, and pour it into a plate.
[0028] Preparation of re-screening medium:
[0029]Egg yolk borax agar plate: NaCl: 0.66%, boric acid: 1.09%, borax: 0.19%, agar: 1.5%, egg yolk liquid: 10%. First weigh 1.32g of NaCl, 2.18g of boric acid, and 0.38g of borax, dissolve them in 180g of water, and adjust the pH to 7.2; add 3g of agar, heat to fully dissolve the agar, add water to the correct volume, and then sterilize. Take 10ml of 50% egg yolk liquid and add it to the borax agar liquid that has been sterilized and cooled to about 55°C, shake well while it is hot, and pour it onto a plate.
Embodiment
[0031] Embodiment: utilize the method for screening of the present invention to produce the PLC strain of the present invention,
[0032] 1. Sampling: Soil samples were collected from Shandong Luhua, Shandong Bohai Grain and Oil, Zhangjiagang Donghai Grain and Oil, Wilmar Shanghai R&D Center, and an oil factory in Taizhou, Zhejiang
[0033] 2. Screen according to the above method to obtain a PLC-producing bacterial strain, which has no lipase activity after analysis.
[0034] 3. Strain identification: Physiological and biochemical characteristics identification, 16S rDNA identification. The result is as follows:
[0035] Table 2 Physiological and biochemical characteristics of bacterial strain P.f-9103 of the present invention
[0036] serial number
Test items
result
[0037] 1
Peptone Water (Tryptophan Broth)
+
2
MR
—
3
VP
—
4
Simon's Citrate
+
5
pyocyanin
—
6
...
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Abstract
The invention relates to a bacterial strain capable of producing phosphatidase C, and a screening method thereof. The bacterial strain is Pseudomonas fluorescens P.f-9103, and preservation number is CCTCC No.M2013298. The bacterial strain is obtained by screening and separation from soil near oil workshops. Morphological characteristics are that: the shape of a bacterial colony is round, the surface of the colony is salient, the color is white, and the bacterial colony is nontransparent and yellows with the increasing of age; the colony is smooth, is relatively viscous and dense, and is easy to be stirred up, and the edge is smooth; the colony has a flagellum, is capable of moving, and produces no blastema; the bacterial strain is gram negative, the optimum growth temperature is 25 to 30 DEG C, and pH value is 7.0 to 7.6. The screening method comprises following steps: primary screening is performed on LB medium containing yolk; then phosphatide is taken as a substrate, and enzymatic reaction is performed; and reaction products are analyzed by HPLC so as to realize secondary screening. According to the method, natural phosphatide is taken as the substrate, so that problems caused by inactivity of phosphatidase C on phosphatide substrate analogue NPPC are avoided. Sources of primary screening raw material are abundant, operation is convenient, secondary screening is accurate and rapid, and the screening method is suitable for high throughput screening of the bacterial strain capable of producing phosphatidase C.
Description
technical field [0001] The invention relates to a strain producing phospholipase C and a screening method thereof, belonging to the field of microorganisms. Background technique [0002] Phospholipase C (PLC, EC3.1.4.3) is a hydrolase that specifically hydrolyzes the phosphatidyl ester bond at the Sn-3 position of natural phospholipids. It plays the role of a second messenger in the life activities of organisms and is widely distributed in bacteria, yeast fungus, slime mold, fruit fly, frog and various mammals, and can be used in the fields of medicine, feed improvement and food industry. In terms of medicine, it is used to develop anti-platelet aggregation drugs to prevent thrombosis and cardiovascular and cerebrovascular diseases. In terms of feed improvement, phospholipase is added to feed to hydrolyze phospholipids to improve feed utilization efficiency and promote animal growth. In the food industry, PLC can be used in oil degumming; at the same time, because phosphol...
Claims
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