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Purification method of plant chlorophyllase
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A chlorophyllase and purification method technology, applied in the direction of hydrolytic enzymes, etc., can solve the problems of complex process, low enzyme yield, excessive enzyme, etc., and achieve the effect of less purification steps and high product purity
Active Publication Date: 2012-12-05
JINGJIANG WEIYI INTPROP SERVICE
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Problems solved by technology
At present, the separation and purification of plant chlorophyllase mainly use methods such as ammonium sulfate fractional precipitation, hydrophobic chromatography, ion exchange, molecular sieve filtration, etc. The process is complicated and the enzyme yield is low.
For example, Tsuchiya et al. (Tsuchiya T, et al. Purification and characterization of two isozymes of chlorophyllase from mature leaves of Chenopodium album. Plant Cell Physiology, 1997, 38 (9): 1026-1031) used ammonium sulfate precipitation in the process of isolating Chenopodium chlorophyllase , ToyopealHW-55 hydrophobic chromatography, Con A chromatography, Heparin chromatography, Mono Q ion exchange chromatography and Superdex 200 molecular sieve filtration 6-step purification to obtain pure chlorophyllase, but too many purification steps cause serious loss of enzyme activity , only one step of Toyopeal HW-55, the enzyme yield is reduced by 91.4%, and the final yield is only 0.24-0.51%
[0004] Shioi et al. (Shioi Y, et al. A simple purification method for the preparation of solublized chlorophyllase from Chlorella protothecoides, Analvtical Biochemistry, 1980, 105(1): 74-79) used ammonium sulfate fractional precipitation, Sepharose CL-6B and Sephadex G-200 Purification of chlorophyllase from Chlorella by molecular sieve chromatography improves the yield of the enzyme and simplifies the purification procedure, but for complex plant cell systems, the purity of the enzyme obtained by this method cannot meet the requirements
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Embodiment 1
[0018] Utilize the method of the present invention to purify Ginkgo chlorophyllase, specifically:
[0019] (1) organic solvent precipitation
[0020] Slowly add acetone to the crude extract of ginkgo leaf protein at 4°C while stirring, the volume percentage concentration of acetone is 40%, centrifuge at 10,000rpm and pour off the supernatant, collect the precipitated part, and add a mass-volume ratio of 0.24% TritonX-100 phosphate buffer (20mmol / L, pH7.0) 10mL, stirred slowly at 4°C for 1h, then centrifuged at 10000rpm, collected the supernatant, discarded the precipitate, the supernatant was Ginkgo chlorophyll enzyme solution 1; The rate of recovery of this step enzyme is 20.5%.
[0021] (2) Ion exchange chromatography
[0022] Enzyme solution I of step (1) was loaded on DEAE-Sephacel, eluted with phosphate buffer (20mmol / L, pH7.0) containing 14.6g / L NaCl, collected components containing chlorophyllase activity, added super Filtrate and concentrate in a centrifuge tube, ce...
Embodiment 2
[0026] Basically the same as Example 1, the difference is that
[0027] The plant material used is bell pepper;
[0028] The organic solvent used in the step (1) is ethanol, and the volume percentage concentration is 30%;
[0029] The ion exchange medium in step (2) is DEAE-Sepharose CL-6B.
Embodiment 3
[0031] Basically the same as Example 1, the difference is that
[0032] The plant material used was pea leaves;
[0033] In step (3), the mass concentration of acrylamide in the stacking gel is 6%, and the mass concentration of acrylamide in the separating gel is 12.5%.
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Abstract
The invention belongs to the technical field of phytobiology, and discloses a purification method of plant chlorophyllase. The method comprises the steps of: taking plant protein coarse extraction liquid as a material; and purifying the chlorophyllase sequentially by an organic solvent precipitation method, an ion exchange chromatography method and a native gel electrophoresis method. The purification method of the plant chlorophyllase is simple in steps, short in period, and convenient to operate, so that the defects of the classical method such as multiple steps, enzyme inactivation and thelike are overcome.
Description
technical field [0001] The present invention relates to a method for purifying plant chlorophyllase, specifically, sequentially adopting organic solvent precipitation, ion exchange chromatography and non-denaturing gel electrophoresis methods to concisely separate and obtain purified chlorophyllase from crude plant protein extract, belonging to field of plant biotechnology. Background technique [0002] Chlorophyllase (Chlorophyllase, EC 3.1.1.14), catalyzes the hydrolysis of chlorophyll to generate dephytyl chlorophyll and phytol. In 1913, Willstatter and Stoll reported the existence of chlorophyllase for the first time in plants. Since then, the research on the physiological function and application of chlorophyllase has been developed rapidly, which can be summarized as: (1) the physiological function of chlorophyllase; (2) the physiological function of chlorophyllase; (3) biochemical kinetics of the enzyme; (4) gene cloning and induced expression; (5) degreening of oil ...
Claims
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