Method for synchronously purifying and immobilizing linoleate isomerase

Abstract

The invention discloses a method for synchronously purifying and immobilizing linoleate isomerase. The method comprises the steps of: treating attapulgite by using hydrochloric acid, adjusting the pH value by using solution of sodium hydroxide, stirring uniformly, filtering, washing, and drying to obtain acid modified attapulgite; mixing the acid modified attapulgite and solution of magnesium sulfate, stirring, filtering, and drying to obtain attapulgite treated by the magnesium sulfate; mixing the attapulgite treated by the magnesium sulfate, gamma-(methacryloyloxy)propyltrimethoxysilane, water and dimethylbenzene and stirring, filtering, washing, and drying to obtain modified attapulgite; mixing enzyme fluid of linoleate isomerase produced from Lactobacillus delbrueckii subsp bulgaricus and the modified attapulgite, stirring, filtering, and drying to obtain the purified and immobilized linoleate isomerase. The method for synchronously purifying and immobilizing the linoleate isomerase is simple and convenient, simple in steps, low in cost, good in purification effect, and high in immobilization efficiency; the activity recovery rate of enzyme reaches over 60 percent during the purification; and the immobilized enzyme has better pH adaptability, thermal stability and operation stability.

Description

technical field [0001] The invention belongs to the field of enzyme purification and immobilization, in particular to a method for simultaneously purifying and immobilizing linoleic acid isomerase. Background technique [0002] Linoleic acid isomerase is usually used to convert linoleic acid into conjugated linoleic acid, and conjugated linoleic acid has many important physiological functions: anti-cancer; anti-oxidation; lowering cholesterol; inhibiting fat accumulation; preventing and treating diabetes; Atherosclerosis; improving bone tissue metabolism; currently, the application potential of conjugated linoleic acid, an essential fatty acid in humans, in medicine, health care, food, feed, etc. has attracted more and more attention. [0003] At present, the production of conjugated linoleic acid mainly adopts chemical methods, such as alkali isomerization of linoleic acid, but the products obtained by chemical methods are a series of CLA mixtures with all positional and ge...

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Problems solved by technology

[0003] At present, the production of conjugated linoleic acid mainly adopts chemical methods, such as alkali isomerization of linoleic acid, but the products obtained by chemical methods are a series of CLA mixtures with all positional and geometric isomers of conjugated double bonds. There are also by-products, but it is very difficult to separate and obtain a single isomer; therefore, more and more attention has been paid to the synthesis of conjugated linoleic acid by biological methods, especially enzymatic methods. However, the nature of the free enzyme is unstable, and it is difficult to recycle the free enzyme after the reaction, which is not conducive to the subsequent separation and purification of the product, and there are many purification steps to obtain the free enzyme, and the yield is low

According to Sun Qi, the master thesis of Northeast Forestry University, Lactobacillus linoleic acid isomerase conversion process of seabuckthorn conjugated linoleic acid, reported the immobilization process of Lactobacillus linoleic acid isomerase, which can be used to prepare immobilized linoleic acid isomerase, but first of all, it needs to centrifuge the fermentation broth, collect the bacteria, ultrasonically combine with lysozyme to break the wall, then ultrasonically treat it twice, extract it with Tritron-100, centrifuge, ammonium sulfate graded salting out, and enzyme solution dialysis , Concentration and other multi-step separation methods to obtain crude enzyme liquid, and then use sodium alginate-calcium chloride embedding method to fix. High energy consumption, cumbersome operation, many steps and many other disadvantages

Patent application number 200610039833.4 discloses a preparation method of immobilized lipase attapulgite. The patent uses purified commercial lipase to make enzyme liquid, and then adopts attapulgite suitable for lipase immobilization. The disadvantage of this method is that the pure enzyme must first be obtained before it can be immobilized

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