Preparation method of chemically modified lipase, lipase and application of lipase in synthesis of L-lactide

A technology for chemically modifying and immobilizing lipase, which is applied in the field of lipase catalysts and can solve problems such as limitations, catalytic activity, stability, and resistance to organic solvents that cannot be effectively improved at the same time

Active Publication Date: 2021-04-23
ANHUI BBCA BIOCHEMICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some research progress has been made in the chemical modification of lipase, it is still limited to the research on common modifiers such as fatty acids, amino acids, acid anhydrides, polyethylene glycol (PEG) and polysaccharides. The catalytic activity, stability, and Properties such as resistance to organic solvents are often not effectively improved at the same time to meet the needs of the reaction

Method used

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  • Preparation method of chemically modified lipase, lipase and application of lipase in synthesis of L-lactide
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  • Preparation method of chemically modified lipase, lipase and application of lipase in synthesis of L-lactide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example provides a chemically modified immobilized lipase. The preparation method comprises: weighing 1 g of lipase (Candida antarctica lipase B, product of Sigma Company), adding 50 mL of sodium phosphate buffer (50 mM, pH7.5) , then add petroleum ether 500mL (which contains 1mL epoxy isopropyl oleate), stir and mix thoroughly for 24h;

[0039] Then add 50 g of solid-phase carrier macroporous resin ECR1030M (Purolite company product), shake the water-bath shaking table for 8 hours (30 ° C, 120 r / min) after adsorption, separate the solid and supernatant by suction filtration, and wash and immobilize with sodium phosphate buffer The enzyme was then vacuum-dried at 35°C for 3 hours to obtain chemically modified and immobilized lipase CALB-M1, which was stored in a refrigerator at 4°C until use.

Embodiment 2

[0044] The lipases prepared in Example 1 and Comparative Example 1 were tested for activity respectively. The determination method is as follows:

[0045] Weigh immobilized lipase (CALB1 and CALB-M1 each 20mg), then add sodium phosphate buffer (50mM, pH7.5) 5mL in a 50mL Erlenmeyer flask, preheat in a constant temperature water bath shaker at 37°C for 15min, then add 5 mL of tributyrin was shaken at 200 rpm for 20 min, and 15 mL of 95% ethanol was added to stop the reaction. Blank is to use buffer instead of enzyme solution. After the reaction was terminated, the fatty acid produced by the reaction was titrated with 0.05 mol / L sodium hydroxide standard solution, using phenolphthalein as indicator.

[0046] Enzyme activity is defined as: under the above conditions, the amount of enzyme required to generate 1 μmol of fatty acid per minute is 1 activity unit, expressed in LU / mg. Calculated as follows:

[0047]

[0048] Among them: V1—the volume of NaOH used for titration, ...

Embodiment 3

[0058] Using chemically modified lipase to catalyze the synthesis of L-lactide, the steps include: the reaction system is carried out in a 1000mL conical flask with a stopper, and the chemically modified immobilized lipase CALB-M1 10.0 prepared in Example 1 is added to the reaction flask g, L-lactic acid 180.0g (2.0mol), 5A molecular sieve 20.0g, petroleum ether 450mL; at 25°C, 400rpm, react for 48h.

[0059] After the reaction, filter and remove 5A molecular sieves, solid-phase enzyme catalysts, etc., and collect the reaction solution; the collected reaction solution is concentrated at 60±2°C in vacuo to remove the solvent petroleum ether, and a light yellow viscous liquid is obtained; the viscous liquid Distill under -0.09MPa vacuum condition, discard the fraction before the low temperature, collect the fraction with a temperature of 130±5°C, cool and crystallize, and obtain 109.4g of L-lactide white crystals, the yield is 76% (109.4 / 180), and the content is 99.5%.

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Abstract

The invention belongs to the technical field of polylactic acid production, particularly discloses chemically modified lipase for synthesizing L-lactide, and further discloses a preparation method of the lipase. According to the preparation method, epoxy isopropyl oleate is adopted for chemically modifying the lipase B derived from Candida antarctica, such that chemically modified lipase is obtained. The chemically modified lipase is high in enzyme activity, and the yield is remarkably improved when the chemically modified lipase is used for catalyzing to generate L-lactide.

Description

technical field [0001] The invention relates to the technical field of polylactic acid production, in particular to the synthesis of lactide, an important intermediate produced by polylactic acid, especially the lipase catalyst used in the synthesis of L-lactide. Background technique [0002] Polylactic acid (referred to as "PLA") is a polymer material with a wide range of uses, and it is also one of the best choices to solve "white pollution". At present, the industry mainly adopts a two-step method to synthesize PLA, and lactide is an important intermediate for the synthesis of PLA. The quality of lactide is one of the key factors affecting the ring-opening polymerization of high molecular weight PLA. The optimization and control of reaction and purification process parameters in the process of lactide synthesis directly determine the quality and yield of lactide, thus affecting the various properties of PLA. [0003] The chemical two-step synthesis of lactide is a relat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C07K1/107C12P17/06
CPCC12N9/20C12Y301/01003C12P17/06
Inventor 李荣杰徐斌汪本助王舒
Owner ANHUI BBCA BIOCHEMICAL CO LTD
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