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Purification method of white gourd prolease

A purification method and protease technology, applied in the field of light industrial food, can solve the problems of high production temperature, inconvenient storage, transportation and production, etc.

Inactive Publication Date: 2008-05-21
惠静璇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production raw materials of these two types of proteases that have been disclosed are all produced in the tropics, and the temperature of the place of production is high, which brings a lot of inconvenience to the storage, transportation and enzyme preparation production of raw materials

Method used

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  • Purification method of white gourd prolease
  • Purification method of white gourd prolease

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Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1: Separation and purification of wax gourd protease

[0018] The first step, the extraction of wax gourd protease: take 1000g of wax gourd, peel it, cut it into small pieces with a fruit knife, put it in an electric homogenizer, and filter the homogenate with 4 layers of gauze, put the filtrate into a centrifuge cup, and keep at 4°C , 10000rpm, centrifuged for 20min. Pour the supernatant into a 1L graduated cylinder to measure the volume and transfer it to a beaker.

[0019] The second step, ammonium sulfate fractional precipitation and dialysis desalination: slowly add ammonium sulfate to the supernatant while stirring to 30% saturation, continue stirring for 30min, 4°C, 10000rpm, centrifuge for 20min, and dissolve the precipitate in 10mLTS buffer solution (20mmol / LTris-Cl, pH8.0, 2mmol / L EDTA, 50mmol / LNaCl), pour the supernatant into a graduated cylinder to measure the volume, continue to add ammonium sulfate to 50% saturation, centrifuge under the same ...

Embodiment 2

[0022] Embodiment 2: Activity measurement and property experiment of wax gourd protease

[0023] 1. Determination of protease activity

[0024] Take 60mL Tris-HCl buffer solution (20mmol / L, pH 8.0) in a small beaker, add 0.75g agar, 1g skimmed milk powder (completely dissolve and then heat to boil, which should be continuously stirred), cool to 50-60 ° C Add 0.6mL of 0.5% streptomycin, shake well and pour into three petri dishes for later use. After solidification, punch some small holes with a diameter of about 5mm with a sterile puncher, and respectively take the purified wax gourd protease samples of the present invention and add them to the holes. In this method, an equal volume of TE buffer or distilled water was used as a control, and incubated at 37°C for 12h. Fig. 2 Determination of casein hydrolysis activity of purified wax gourd protease, 1: wax gourd juice (5 μL); 2: wax gourd juice (15 μL); 3: protein eluted from DEAE-Sepharose FF ion exchange column (2 μ g); 4 :...

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Abstract

The invention relates to a purification method of wax gourd proteinase. The wax gourd is grinded, filtered and centrifuged; the supernatant is precipitated by ammonium sulfate with various saturation to obtain protein precipitation which is dissolved by TS buffer; through dialysis and DEAE-Sepharose FF ion exchange column gradient elution, 0.2mol / L NaCl eluate is collected for concentration and Superdex 75 gel filtration chromatography; the purified wax gourd proteinase with 65KD to 67KD molecular weight. The proteinase has significant casein hydrolysis activity; the best pH is 6.2 to 6.6 and the best temperature is 68 degree to 72 degree; the invention has good thermal stability; the proteinase still preserves 90 percent activity at 80 degree for 100 minutes. The invention can be applied in areas such as food processing, textile processing and medical healthcare etc.

Description

Technical field: [0001] The invention relates to a method for purifying wax gourd protease, which belongs to the field of light industrial food. Background technique: [0002] Winter melon (Benincasa hispida Cogn.), also known as white melon, pillow melon, wide melon, originates in southern my country and India, belongs to Cucurbitaceae, and is cultivated all over the country. Winter melon is rich in nutrition, storage-resistant, white in texture, crisp and juicy, and has diuretic, heat-clearing, phlegm-resolving, thirst-quenching effects. [0003] Protease is a kind of enzyme that specifically hydrolyzes protein. Because of its specific substrate and high catalytic efficiency, it has been widely used in medicine, food, chemical industry, feed, textile and other fields. It is one of the few industrialized products. One of the enzyme preparations. The raw materials of protease preparation mainly include animals, plants and microorganisms, and microorganisms are the main one...

Claims

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Application Information

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IPC IPC(8): C12N9/50
Inventor 惠静璇
Owner 惠静璇
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