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Method for rapidly extracting cell wall acid invertase in fruit pulp
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A technology of acid inverting enzymes and cell walls, applied in the field of biochemistry, to achieve the effects of improving test efficiency, simple operation, and shortening test time
Active Publication Date: 2018-02-06
SUZHOU UNIV
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At present, there is no report on the use of ultrasonic treat
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Embodiment 1
[0016] 1. To prepare extraction buffer and elution buffer:
[0017] Extraction buffer Ⅰ is 150 mm Tris-HCl solution (pH 8.0), 10 mm MgCl 2 , 2 mm EDTA, 0.1 mmPMSF, 0.2% (v / v) β-mercaptoethanol, 1 mm benzamidine, 3% (w / v) PVPP and 10 mM ascorbic acid;
[0018] Extraction buffer II is 150 mm Tris-HCl solution (pH 8.0), 10 mm MgCl 2 , 2 mM EDTA, 0.1 mmPMSF, 0.2% (v / v) β-mercaptoethanol, 1 mM benzamidine, and 10 mM ascorbic acid;
[0019] Elution buffer was 150 mm Tris-HCl solution (pH 8.0), 10 mm MgCl 2 , 2 mM EDTA, 0.1 mmPMSF, 0.2% (v / v) β-mercaptoethanol, 1 mM benzamidine, and 10 mM ascorbic acid; 0.5 m NaCl.
[0020] 2. Weigh 1 g of loquat pulp sample, freeze and grind in liquid nitrogen, add 6 mL of pre-cooled extraction buffer, 12,000× g After refrigerated centrifugation for 30 min, the supernatant was removed to obtain a precipitate.
[0021] 3. Repeatedly wash the precipitate 4-5 times with extract solution II, each time with 6 ml, gently shake the centrifuge tube ...
Embodiment 2
[0025] 1. adopt prior art to extract cell wall invertase, its method is as follows:
[0026] (1) Extraction buffer and elution buffer are the same as Step 1 in Example 1.
[0027] (2) Weigh an appropriate amount of loquat pulp, freeze and grind in liquid nitrogen, add pre-cooled extraction buffer Ⅰ, 12,000× g After refrigerated centrifugation for 30 min, the supernatant was removed to obtain a precipitate.
[0028] (3) Repeatedly wash the precipitate several times with PVPP-free extraction solution II, shake the centrifuge tube gently to dissolve and suspend the precipitate in the washing solution, and then centrifuge to obtain the precipitate and supernatant (washed out washing solution); Then use Coomassie Brilliant Blue G250 to detect the washing solution obtained by washing until the washing solution does not contain protein.
[0029] (4) The washed precipitate was then eluted with extraction solution II containing 0.5 m NaCl but no PVPP, shaken gently for 24 hours, and ...
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Abstract
The invention discloses a method for rapidly extracting cell wall acid invertase in fruit pulp. The method comprises the following steps: with the fruit pulp as the raw material, grinding with liquidnitrogen, adding a pre-cooled extracting buffer solution, carrying out centrifugation so as to obtain precipitates, repeatedly washing the precipitates by virtue of washing liquid so as to remove soluble proteins, adding an eluent into the washed precipitates, carrying out ultrasonic treatment, and maintaining the wate-bath temperature in the whole ultrasonic treatment process at 0-4 DEG C; and after the ultrasonic treatment is finished, centrifuging the obtained supernate, so as to obtain a crude enzyme extracting solution of the cell wall acid invertase. The activity of the cell wall acid invertase in the obtained extracting solution has no remarkable difference with that of the cell wall acid invertase extracted through a conventional 24h vibration manner; and furthermore, the method issimple, convenient, rapid, low in consumption and suitable for extracting the cell wall acid invertase in various fruits, and the obtained extracting solution can be applied to the activity analysisof the cell wall acid invertase.
Description
technical field [0001] The invention relates to a method for extracting cell wall acid invertase in fruits, belonging to the field of biochemistry. Background technique [0002] The content of sugar in the fruit, the type of components and the proportion of each component are the key factors to determine the sweetness and flavor quality of the fruit. It is also the basic raw material for the synthesis of carotenoids, vitamins, pigments and aromatic substances. Supply, and play the role of signaling molecules in cell transduction. Sucrose is an important factor determining the sweetness of fruit, and the accumulation of sucrose in fruit is related to the activity of related metabolic enzymes. Sucrose invertase exists widely in fruits and can be divided into three types according to their pH value and subcellular localization: soluble acid invertase (S-AIV), neutral invertase (NINV) and cell wall invertase (CWINV). Cell wall invertase (CWINV), an acid-insoluble invertase, is...
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