Macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and method

A technology of papain and covalent cross-linking, which is applied in the field of preparation of new immobilized enzyme biocatalysts, can solve the problems of chitosan carrier instability, unfavorable repeated recycling, low enzyme loading, etc., to overcome the unfavorable industrial Application, improvement of industrialization defects, simple preparation method

Inactive Publication Date: 2011-09-14
TIANJIN UNIV
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Problems solved by technology

Li Hong discloses a kind of chitosan microsphere is prepared as raw material with chitosan, chitosan microsphere is added in the papain solution, obtains the papain that chitosan microsphere immobilizes through adsorption-crosslinking again (application number : 00117351.0), the selected chitosan carrier in this method is unstable in acidic solution, which easily causes papain to come off from the carrier
[00

Method used

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  • Macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and method
  • Macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and method
  • Macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and method

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Experimental program
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Embodiment 1

[0037]Take 2g of macroporous silica gel (average pore diameter 1 μm, average particle diameter of carrier particles 5 mm, average porosity 0.6), wash and dry at 100° C. for 12 h until the mass does not change. According to the mass ratio of macroporous silica gel to silane coupling agent volume ratio of 1:5 (g:mL), take 10mL of γ-aminopropyltriethoxysilane and 20mL of absolute ethanol and mix it for 10min, then add 2g of dried macroporous silica gel carrier. Rotary evaporation under reduced pressure at 50°C until the liquid was no longer reduced, the carrier was washed three times with absolute ethanol, and dried at 100°C to obtain an amino-modified macroporous silica gel carrier, the surface amino content was measured to be 8.5 μmol / g. Dissolve papain in 0.2mol / L phosphate buffer (pH7.0) to prepare 4mL of 0.15g / mL enzyme solution, add 2g of amino-modified macroporous silica gel carrier, and shake at 25°C for 3.5h . The carrier adsorbed with papain was taken out, washed once...

Embodiment 2

[0039] Take 6g of macroporous aluminum oxide (average pore diameter 0.8 μm, average particle diameter of carrier particles 5 mm, average porosity 0.5), wash it, and dry it at 120° C. for 12 hours until the mass does not change. Take 20mL of N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane and mix it with 60mL of absolute ethanol for 20min according to the mass ratio of macroporous silica gel to silane coupling agent volume ratio of 1:3.3 (g:mL). Finally, 6 g of dried macroporous aluminum oxide carrier was added. Rotary steam under reduced pressure at 60°C until the liquid no longer decreases, wash the carrier four times with absolute ethanol, and dry at 120°C to obtain an amino-modified macroporous alumina carrier, the surface amino content is measured to be 10 μmol / g . Dissolve papain in 0.1 mol / L phosphate buffer (pH 4.0) to prepare 3 mL of 0.25 g / mL enzyme solution, add 3 g of amino-modified macroporous alumina carrier, shake at 4 °C Shake for 6 hours. The carrier adsorbed...

Embodiment 3

[0041] Take 10 g of macroporous cellulose particles (average pore diameter 1 μm, average carrier particle diameter 5 mm, average porosity 0.6), wash them, and dry them at 90° C. for 24 h until the mass does not change. Take 30mL of N-β-(aminoethyl)-γ-aminopropylmethyldimethoxysilane and mix it with 30mL of absolute ethanol according to the mass ratio of macroporous silica gel to silane coupling agent volume ratio of 1:3 (g:mL). After 30 minutes, 10 g of dried macroporous cellulose carrier was added. Rotary evaporation under reduced pressure at 70°C until the liquid was no longer reduced, the carrier was washed three times with absolute ethanol, and dried at 90°C to obtain an amino-modified macroporous cellulose carrier. The surface amino content was measured to be 8.7 μmol / g. Dissolve papain in 0.2mol / L phosphate buffer (pH10.0) to prepare 5mL of 0.05g / mL enzyme solution, add 5g of amino-modified macroporous cellulose carrier, shake at 15°C for 5h . The carrier adsorbed with...

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Abstract

The invention relates to a macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and a method. In the papain polymer, a papain is embedded in the pore canal of a carrier the aperture of which is more than 0.5mu m; an inorganic macroporous material or an organic macroporous material the surface of which is provided with hydroxyl is taken as an immobilized enzyme carrier; synchronous complementation of the formation of the crosslinking papain polymer and the covalent connection of the crosslinking papain polymer and the macroporous carrier is finally realized through amino-group modification, enzyme adsorption, enzyme precipitation and synchronous crosslinking on the surface of the carrier; the enzyme carrying amout of the immobilized enzyme is higher than that of the immobilized enzyme of a common carrier; the enzyme leakage caused by applications of the macroporous carrier is avoided by adopting a covalent fixed method; the carrier shape can be adjusted according to the actual requirements; and the optimum catalysis temperature, pH value, solvent and heat stability are obviously improved. The papain polymer provided by the invention can be applied to other proteases, lipase, amylase, glucose isomerase, penicillin, acylase, pectinase, oxidase, L-asparaginase, aspartase and peroxidase and the like.

Description

technical field [0001] The invention relates to a preparation method of a novel immobilized enzyme biocatalyst, in particular to a "synchronous method" covalent cross-linking-immobilized papain polymer and method using a macroporous material as a carrier, belonging to the technical field of enzyme immobilization . Background technique [0002] Cross-linked enzyme aggregates (cross-linked enzyme aggregates, CLEAs) are a new method of immobilizing enzymes. In the preparation process, firstly, a precipitant is used to aggregate and precipitate dissolved enzymes, and then the enzyme-precipitated aggregates are covalently cross-linked. couplet. The immobilized enzyme prepared by the method has a wide optimum catalytic pH range and a wide optimum catalytic temperature range, and has high stability to high temperature, organic solvent, strong acid and strong alkali, etc., and is an efficient, stable immobilized enzyme method. However, CLEAs have relatively low mechanical strengt...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/12
Inventor 齐崴王梦凡苏荣欣何志敏
Owner TIANJIN UNIV
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