Preparation method of biomineralized immobilized lipase and application of biomineralized immobilized lipase in catalytic synthesis of OPO

A technology for immobilizing lipase and fat, applied to biochemical equipment and methods, enzymes immobilized on or in inorganic carriers, etc., can solve the problems of poor operation and storage stability of natural enzymes, difficulties in enzyme recovery and reuse, Impact and other issues

Inactive Publication Date: 2020-09-11
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, natural enzymes generally have poor operation and storage stability in practical applications, difficult recovery and reuse of enzymes, high cost, and are also affected by the reaction solution.

Method used

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  • Preparation method of biomineralized immobilized lipase and application of biomineralized immobilized lipase in catalytic synthesis of OPO
  • Preparation method of biomineralized immobilized lipase and application of biomineralized immobilized lipase in catalytic synthesis of OPO
  • Preparation method of biomineralized immobilized lipase and application of biomineralized immobilized lipase in catalytic synthesis of OPO

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Experimental program
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Effect test

Embodiment 1

[0030] refer to figure 1 , the lipase TLL was immobilized by biomineralization, the lipase was dispersed in phosphate buffer, and a certain proportion of CaCl was added 2 solution, a white precipitate was formed, and then the mixture was left standing at 4°C to obtain immobilized lipase, which was used to catalyze the synthesis of 1,3-dioleyl-2-palmitoylglycerol (OPO).

[0031] In this invention, a simple method was used to co-produce TLL-hydroxyapatite nanoflowers (TLL@HAp-NFs) complexes with calcium phosphate through a biomineralization process, and it was applied to the synthesis of OPO, first , TLL gene was constructed on the pPIC9K vector, and then the recombinant plasmid was transferred to Pichia pastoris strain GS115, and lipase TLL was obtained after induced expression.

[0032] Preparation of TLL@hydroxyapatite nanoflowers (TLL@HAp-NFs)

[0033] First, the concentration of the purified and concentrated lipase protein was detected by the Bradford protein detection kit,...

Embodiment 2

[0038] Synthesis of OPO Catalyzed by TLL@Hydroxyapatite Nanoflowers

[0039] TLL@HAp-NFs were used as biocatalysts for the enzymatic acid hydrolysis of tripalmitin (PPP) and oleic acid (OA) to produce OPO. The effects of different reaction conditions on the product were studied, including water content, reaction temperature, molar ratio of reactants and so on.

[0040] The catalytic transesterification reaction is as follows: PPP (0.24mmol), OA (0.48-2.40mmol), TLL@hydroxyapatite nanoflowers (5-25mg·mL -1 ) into different solvents (6mL total volume, n-pentane, n-hexane, n-heptane, n-octane, cyclohexane and acetone) to dissolve, the water content in the reaction is 2%, the reaction temperature is 25-55°C, and The speed of 220rpm was reacted in the shaker for 12h. Solvents such as n-hexane used in the reaction need to use molecular sieves Dry for 24+ hours. Take 500 μL of each sample, and then add 500 μL of chromatographic grade n-hexane to mix. All experiments were repeat...

Embodiment 3

[0042] Effect of Reaction Temperature on Catalytic Reaction

[0043] The catalytic results at reaction temperatures of 25°C, 35°C, 45°C and 55°C were mainly explored and compared. In this experiment, we found that not all reactions increased yield with increasing temperature, but free enzyme TTL was more sensitive to increasing temperature. At 25°C and 35°C, the OPO content in the reaction catalyzed by the free enzyme can be kept above 40%, but at 45°C and 55°C, the OPO content decreases significantly. However, the change of OPO content in the catalytic reaction solution of TLL@hydroxyapatite nanoflowers is relatively stable, and the OPO content is at least 35%. But too high a temperature can also negatively affect the immobilized enzyme. Analysis of its reason may be that the temperature is too high to affect the activity of the enzyme, and the temperature is too low will cause the substrate tripalmitin can not be completely dissolved in the reaction solvent, so that the en...

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Abstract

The invention relates to a preparation method of biomineralized immobilized lipase and application of the biomineralized immobilized lipase in catalytic synthesis of OPO. The method comprises the following steps: adding lipase and a certain proportion of calcium chloride solution into a phosphate buffer solution, then standing a mixture at 1-8 DEG C to obtain immobilized lipase, and applying the immobilized lipase to catalytic synthesis of 1,3-dioleoyl-2-palmitoyl glyceride. The process is simple, the temperature stability of the prepared immobilized lipase is obviously improved, and when theimmobilized lipase is used for catalytic synthesis of the 1,3-dioleoyl-2-palmitoyl glyceride, the catalytic yield is high; and according to the invention, the thermal stability and catalytic activityof the lipase can be effectively improved.

Description

technical field [0001] The invention relates to an enzyme immobilization method, in particular to the preparation of a biomineralization immobilized lipase and its application in catalyzing the synthesis of OPO. Background technique [0002] Enzymes are proteins that catalyze biological and chemical reactions and are widely found in plants, animals and microorganisms. It has the advantages of high catalytic activity, strong selectivity, good specificity, low cost, mild reaction conditions and biodegradability. Therefore, enzymes have high applications in various fields such as fine chemistry, medicinal chemistry, food or energy. Biocatalytic processes have received extensive attention as a rational and efficient conversion method. Enzymatic processes play an important role in supporting sustainable green chemistry. However, natural enzymes generally have poor operation and storage stability in practical applications, difficult recovery and reuse of enzymes, high cost, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12P7/64
CPCC12N9/20C12N11/14C12P7/6445C12Y301/01003
Inventor 谢恬王安明李宁宁张静
Owner HANGZHOU NORMAL UNIVERSITY
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