CN107663519B - Method for rapidly extracting cell wall acid invertase in fruit pulp - Google Patents
Method for rapidly extracting cell wall acid invertase in fruit pulp Download PDFInfo
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- CN107663519B CN107663519B CN201710995300.1A CN201710995300A CN107663519B CN 107663519 B CN107663519 B CN 107663519B CN 201710995300 A CN201710995300 A CN 201710995300A CN 107663519 B CN107663519 B CN 107663519B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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Abstract
The invention discloses a method for rapidly extracting cell wall acid invertase in fruit pulp. Taking fruit pulp as a raw material, grinding the fruit pulp by using liquid nitrogen, adding a pre-cooled extraction buffer solution, repeatedly washing precipitates obtained after centrifugation by using a washing solution for many times to remove soluble protein, adding an eluent into the precipitates obtained after washing, performing ultrasonic treatment, and keeping the water bath temperature of 0-4 ℃ in the whole ultrasonic treatment process. And after the ultrasonic treatment is finished, centrifuging to obtain supernatant, namely the crude enzyme extracting solution of the cell wall acid invertase. The activity of the cell wall acid invertase in the extracting solution extracted by the method has no obvious difference with the activity of the cell wall acid invertase extracted by conventional 24-hour oscillation, and the method is simple, convenient, quick and low in consumption, is suitable for extracting the cell wall acid invertase in various fruits, and can be used for analyzing the activity of the cell wall acid invertase.
Description
Technical Field
The invention relates to a method for extracting cell wall acid invertase in fruits, belonging to the field of biochemistry.
Background
The content of sugar in the fruit, the types of components and the proportion of each component are key factors for determining the sweetness and the flavor quality of the fruit, are basic raw materials for synthesizing carotenoids, vitamins, pigments, aromatic substances and the like, participate in metabolism and energy supply, and play a role of signal molecules in cell transduction. Sucrose is an important factor for determining the sweetness of fruits, and the accumulation of sucrose in fruits is related to the activity of its associated metabolic enzymes. Sucrose invertase is widely present in fruits and is classified into 3 types according to its pH and subcellular localization: soluble acid invertase (S-AIV), Neutral Invertase (NINV) and Cell Wall Invertase (CWINV). Cell Wall Invertase (CWINV) is an insoluble acid invertase, considered as a key enzyme for sucrose unloading; the enzyme plays an important role in the transport of photosynthetic products by maintaining a sucrose concentration gradient between sucrose and sink tissues. The common extraction method of the cell wall invertase adopts long-time (24 h) oscillation treatment, so that the cell wall invertase is extracted from the cell wall, not only is the time consumed, but also the low-temperature process needs to be kept for a long time, and the energy consumption is large.
Ultrasonic wave means frequency of 2 × 104—2×109The sound wave in Hz is an elastic mechanical vibration above the normal human hearing range. The ultrasonic wave is widely applied to food, medical treatment and health and raw food after being firstly generated in 1830 yearsMany fields such as extraction of substance molecules; the ultrasonic extraction can realize the process of breaking cell walls and efficiently and quickly extracting the inner solute in the cells. The ultrasonic extraction is a method for increasing the molecular motion frequency and speed and increasing the penetrating power of a solvent by utilizing the cavitation, mechanical action, thermal effect and the like of ultrasonic waves, thereby improving the leaching rate of target components. The method has the advantages of time saving, high extraction efficiency and the like, and is a rapid and efficient extraction method. At present, no report is found about the application of ultrasonic treatment technology to the extraction of cell wall acid invertase in fruits.
Disclosure of Invention
The invention provides a method for quickly, simply and conveniently extracting fruit cell wall acid invertase aiming at the defect of overlong extraction time of the cell wall invertase at present, and provides a crude enzyme extracting solution for measuring the activity of the cell wall invertase.
In order to achieve the above purpose, the technical scheme adopted by the invention is to provide a method for rapidly extracting fruit cell wall invertase, which comprises the following steps at the temperature of 0-4 ℃:
1. preparing extraction buffer solutions I and II and an elution buffer solution respectively:
the extraction buffer I was 150 mm Tris-HCl pH 8.0, 10 mm MgCl22 mm EDTA, 0.1 mm PMSF, 0.2% v/v beta-mercaptoethanol, 1 mm benzamidine, 3% w/v PVPP and 10 mm ascorbic acid;
the extraction buffer II was 150 mm Tris-HCl pH 8.0, 10 mm MgCl22 mm EDTA, 0.1 mm PMSF, 0.2% v/v beta-mercaptoethanol, 1 mm benzamidine, 10 mm ascorbic acid;
the elution buffer was 150 mm Tris-HCl pH 8.0, 0.5 m NaCl, 10 mm MgCl22 mm EDTA, 0.1 mm PMSF, 0.2% v/v beta-mercaptoethanol, 1 mm benzamidine, 10 mm ascorbic acid;
2. grinding a fruit pulp sample, adding an extraction buffer solution I, freezing and centrifuging, and removing a supernatant to obtain a precipitate;
3. repeatedly washing the precipitate by using an extraction buffer solution II to remove protein;
4. and (3) adding an elution buffer solution into the precipitate obtained in the step (3), carrying out ultrasonic extraction for 2-3 h under the conditions that the ultrasonic frequency is 40KHZ and the power is 100W, and then carrying out centrifugal treatment to obtain a supernatant, namely a crude enzyme extracting solution of the cell wall invertase in the fruit pulp.
Compared with the prior art, the invention has the following advantages: the invention utilizes the advantage of ultrasonic extraction of biomacromolecules, applies the ultrasonic extraction technology to the extraction of fruit cell wall invertase, can lead the extraction to be rapid, and proves that the activity of the cell wall invertase in the crude enzyme liquid obtained by 3h ultrasonic extraction has no obvious difference with the activity of the cell wall invertase in the crude enzyme liquid obtained by 24h oscillation through comparative tests and enzyme activity detection. The method for extracting the fruit cell wall invertase by using the ultrasonic waves can greatly shorten the test time, is simple to operate and can effectively improve the test efficiency.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following specific examples.
Example 1
1. Preparing an extraction buffer solution and an elution buffer solution:
the extraction buffer I was 150 mm Tris-HCl solution (pH 8.0), 10 mm MgCl22 mm EDTA, 0.1 mm PMSF, 0.2% (v/v) beta-mercaptoethanol, 1 mm benzamidine, 3% (w/v) PVPP and 10 mm ascorbic acid;
the extraction buffer II was 150 mm Tris-HCl solution (pH 8.0), 10 mm MgCl22 mm EDTA, 0.1 mm PMSF, 0.2% (v/v) beta-mercaptoethanol, 1 mm benzamidine and 10 mm ascorbic acid;
the elution buffer was 150 mm Tris-HCl solution (pH 8.0), 10 mm MgCl22 mm EDTA, 0.1 mm PMSF, 0.2% (v/v) beta-mercaptoethanol, 1 mm benzamidine and 10 mm ascorbic acid; 0.5 m NaCl.
2. Weighing 1 g of loquat pulp sample, freezing and grinding the loquat pulp sample by using liquid nitrogen, adding 6 mL of precooled extraction buffer solution, 12,000gFreezing and centrifuging for 30 min, and removing supernatant to obtain precipitate.
3. Repeatedly washing the precipitate with the extracting solution II for 4-5 times, wherein the amount of the extracting solution is 6 ml each time, slightly shaking the centrifugal tube to dissolve and suspend the precipitate in the washing solution, and then centrifuging to obtain the precipitate and a supernatant (washed washing solution); the washed-out wash was then checked with Coomassie Brilliant blue G250 until the eluate was free of protein.
4. Adding 2 ml of elution buffer solution into the sediment obtained after washing, putting the centrifuge tube into a laboratory ultrasonic cleaner, performing ultrasonic extraction for 3 hours (ensuring the water temperature in the ultrasonic cleaner to be 0-4 ℃ by adding ice cubes in the ultrasonic extraction process), and then performing 12,000-fold extraction on the sedimentg Freezing and centrifuging for 30 min to obtain supernatant as crude enzyme extract of CWINV.
The whole extraction process is carried out at a low temperature of 0-4 ℃.
Example 2
1. The method for extracting the cell wall invertase by adopting the prior art comprises the following steps:
(1) the extraction buffer and elution buffer were the same as in step 1 of example 1.
(2) Weighing a proper amount of loquat pulp sample, freezing and grinding the loquat pulp sample by using liquid nitrogen, and adding precooled extraction buffer solution I, 12,000gFreezing and centrifuging for 30 min, and removing supernatant to obtain precipitate.
(3) Repeatedly washing the precipitate with an extracting solution II without PVPP for several times, slightly shaking the centrifuge tube to dissolve and suspend the precipitate in the washing solution, and then centrifuging to obtain the precipitate and a supernatant (washed washing solution); the washed-out wash was then checked with Coomassie Brilliant blue G250 until the eluate was free of protein.
(4) And eluting the washed precipitate with an extracting solution II containing 0.5 m NaCl but not containing PVPP, slightly shaking for 24h, and centrifuging for 30 min to obtain a supernatant which is a crude enzyme extracting solution of CWINV.
The whole extraction process is carried out at a low temperature of 0-4 ℃.
The crude CWIV enzyme extract obtained by the above method is used as a comparative example.
Determination of CWINV Activity
The crude enzyme solutions extracted in example 1 and comparative example were each subjected to measurement of cell wall-converting enzyme activity. The determination method comprises the following steps: the CWIV activity determination method is slightly modified. The total volume of the reaction was 1 mL, including: 30 mM acetic acid-potassium acetate (pH = 4.5), 50 μ L enzyme extract and 100 mM sucrose. The mixed solution is kept warm for 30 min in a water bath at 37 ℃, then 1 mL of DNS is added to stop the reaction, and the mixture is boiled in the water bath for 5 min. The amount of reducing sugars released by sucrose was determined at 540 nm after cooling. Enzyme blank was used as control and substrate blank as reference. The enzyme blank reaction system only contains enzyme extract and reaction liquid and does not contain a substrate; the substrate blank reaction system contained only the substrate and the reaction solution, and did not contain the enzyme solution (Tanase and Yamaki, 2000).
Enzyme activity calculation formula: enzyme Activity (μmol. h)-1g-1FW) = [ sucrose or glucose content (μ g) calculated according to standard curve/molecular weight of sucrose or glucose/1 mL × total volume of enzyme extract (mL)]/[ fresh weight of sample (g) × volume of enzyme solution at reaction (mL)/total volume of reaction solution (mL) × reaction time (h)]
3. Comparison of measurement results
The activity of the cell wall-converting enzyme in the enzyme solution obtained by the ultrasonic extraction method of example 1 was
42.00 μmol·h-1·g-1FW, comparative example cell wall converting enzyme activity determined by conventional method was 40.31. mu. mol. h-1·g-1FW. The SPSS 17.0 is used for carrying out difference significance analysis, and the result shows that the two activity values have no significant difference.
The results show that the activity of the cell wall acid invertase in the extracting solution extracted by the invention has no significant difference from the activity of the cell wall acid invertase extracted by conventional 24-hour oscillation. The method is simple, rapid, low in consumption, and suitable for extracting cell wall acid invertase from various fruits, and the obtained extractive solution can be used for analyzing cell wall acid invertase activity.
Claims (1)
1. A method for rapidly extracting cell wall acid invertase from fruit pulp is characterized by comprising the following steps of:
(1) preparing extraction buffer solutions I and II and an elution buffer solution respectively:
the extraction buffer I was 150 mM Tris-HCl pH 8.0, 10 mM MgCl22 mM EDTA, 0.1 mM PMSF, 0.2% (v/v) β -mercaptoethanol, 1 mM benzamidine, 3% (w/v) PVPP and 10 mM ascorbic acid;
the extraction buffer II was 150 mM Tris-HCl pH 8.0, 10 mM MgCl22 mM EDTA, 0.1 mM PMSF, 0.2% (v/v) β -mercaptoethanol, 1 mM benzamidine, 10 mM ascorbic acid;
the elution buffer was 150 mM Tris-HCl pH 8.0, 0.5M NaCl, 10 mM MgCl22 mM EDTA, 0.1 mM PMSF, 0.2% (v/v) β -mercaptoethanol, 1 mM benzamidine, 10 mM ascorbic acid;
(2) grinding a fruit pulp sample, adding an extraction buffer solution I, freezing and centrifuging, and removing a supernatant to obtain a precipitate;
(3) repeatedly washing the precipitate by using an extraction buffer solution II to remove protein;
(4) adding an elution buffer solution into the precipitate obtained in the step (3), performing ultrasonic extraction for 2-3 h under the conditions that the ultrasonic frequency is 40KHZ and the power is 100W, and then performing centrifugal treatment to obtain a supernatant, namely a crude enzyme extracting solution of the cell wall invertase in the fruit pulp.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676474A (en) * | 2012-06-01 | 2012-09-19 | 南京农业大学 | Method for extracting pear sucrose invertin and method for measuring activity thereof |
| CN102747055A (en) * | 2012-06-19 | 2012-10-24 | 南京农业大学 | Method for extracting sucrose synthase and sucrose phosphate synthase of pear fruit and activity determination method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676474A (en) * | 2012-06-01 | 2012-09-19 | 南京农业大学 | Method for extracting pear sucrose invertin and method for measuring activity thereof |
| CN102747055A (en) * | 2012-06-19 | 2012-10-24 | 南京农业大学 | Method for extracting sucrose synthase and sucrose phosphate synthase of pear fruit and activity determination method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 植物细胞壁蛋白质提取方法的改进;陈绍宁等;《河南农业大学学报》;20091015;第45卷(第2期);第557-558页材料与方法1.2-1.3，第558页结果与分析2.1 * |
| 橡胶树叶片转化酶提取方法的比较;何慧等;《热带作物学报》;20120325;第33卷(第3期);第494页1.2.1-1.2.2 * |
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