Immobilized lipase as well as preparation method and application thereof in biodiesel

A technology of immobilized lipase and lipase, applied in the direction of immobilized on/in organic carriers, biochemical equipment and methods, biofuels, etc., can solve the problems of high acid value, long reaction time, and poor stability of biodiesel problem, achieve the effect of simple preparation method, low cost and good operation stability

Active Publication Date: 2020-11-17
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of long reaction time, high acid value of biodiesel and poor enzyme stability in the current enzymatic preparation of biodiesel, the invention provides an immobilized lipase and its application technology for preparing biodiesel

Method used

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  • Immobilized lipase as well as preparation method and application thereof in biodiesel
  • Immobilized lipase as well as preparation method and application thereof in biodiesel
  • Immobilized lipase as well as preparation method and application thereof in biodiesel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 prepares lipase MAS1-H108A

[0050] The preparation process of lipase MAS1-H108A is as follows:

[0051] (1) Activation of strains: Linearize the MAS1-H108A-pPICZαA-PDI plasmid, electroporate it into Pichia pastoris X33 competent, and screen out positive strains; Streak the Pichia pastoris X33 glycerol strain on YPD containing Zeocin Solid plate, cultivated in an incubator at 30°C for 60-75h;

[0052] (2) Preparation of primary seed liquid: inoculate the obtained single colony into a sterilized Erlenmeyer flask containing 50mL of YPD liquid medium, then place the Erlenmeyer flask at a temperature of 30°C and a shaking speed of 200rpm Shake culture in the bed for 18-24 hours to obtain the first-grade seed solution;

[0053] (3) Preparation of the secondary seed solution: Inject 5 mL of the primary seed solution into 100 mL of sterilized fresh YPD liquid medium, and shake and cultivate it in a shaker at 30° C. at a rotation speed of 200 rpm for 24 hours;

...

Embodiment 2

[0056] Example 2 Preparation of immobilized lipase MAS1-H108A

[0057] Lipase MAS1-H108A and resin D5753 (pore size 200-350nm, ultra-large pore size, purchased from Purite Company), Lifetech TM ECR1030M (pore diameter 22-34nm, mesoporous resin, purchased from Brilliant Company), Lifetech TM ECR8285 (aperture 45-65nm, macroporous resin, purchased from Brilliant Company), AMBERLITE TM XAD TM 1180N (pore diameter 30-55nm, mesoporous resin, purchased from Dow Company), AB-8 (pore diameter 13-14nm, mesoporous resin, purchased from Tianjin Jinda Resin Factory) was immobilized. After the fermentation broth was centrifuged and concentrated, 20 mL of phosphate buffer was added to 100 mL of enzyme liquid to adjust the pH to 7; and the carrier was added according to the ratio of 10,000 U / g of total enzyme activity / carrier, and mixed with the enzyme liquid for 8 hours. The immobilized enzyme was suction filtered using a Buchner funnel, the immobilized enzyme was washed with phosphate b...

Embodiment 3

[0059] Example 3 Preparation of immobilized enzymes using resin D5753 and different lipases

[0060] Five lipases were selected: lipase MAS1-H108A (abbreviated as MAS1), CALB (derived from Candidaantarctica), Eversa Transform 2.0 (abbreviated as ET), RML (derived from Rhizomucor miehie), TLL (derived from Thermomyces lanuginosus), and Resin D5753 (pore size 200-350nm, ultra-large pore size) was combined to prepare 5 kinds of immobilized enzymes. The immobilization method is to add 20% phosphate buffer solution to the enzyme solution, adjust the pH to 8; add the carrier according to the ratio of 10,000U / g total enzyme activity / carrier, mix and stir with the enzyme solution for 8 hours, and use the Buchner funnel to immobilize Suction filter the immobilized enzyme, wash the immobilized enzyme with phosphate buffer until no protein exists in the filtrate, drain the immobilized enzyme and dry it at 40°C for 6 hours. Immobilized lipase can be prepared.

[0061] The esterification...

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Abstract

The invention discloses an immobilized lipase as well as a preparation method and application thereof in biodiesel. A lipase MAS1-H108A and a carrier are immobilized, and the amino acid sequence of the lipase MAS1-H108A is as shown in SEQ NO.1; and the carrier is hydrophobic non-polar resin, and the pore diameter of the carrier is greater than or equal to 30 nm. The immobilized enzyme has high esterification capacity and high transesterification capacity at the same time, resists high temperature and organic solvents, retains higher catalytic capacity after being reused for multiple times, andis suitable for preparing biodiesel. By using the immobilized lipase, the biodiesel can be efficiently prepared at the temperature of 20-55 DEG C, the yield of the biodiesel is 95-98%, the acid valueis 0.7-2 mg/g, and the catalytic effect after 30-50 batches of reaction is not obviously reduced, so that the problems of long time, high acid value of the biodiesel and poor enzyme stability in an enzymatic reaction process are solved to a certain extent.

Description

technical field [0001] The present invention relates to containing immobilized lipase and its preparation method. The invention also relates to the application of the immobilized lipase in the production of biodiesel. Background technique [0002] In recent years, fossil energy has been depleted and the ecological environment has been deteriorating. The development of renewable energy is a strategic choice for the country to achieve sustainable development and low-carbon economy. Biodiesel refers to fatty acid monoalkyl esters, such as fatty acid methyl esters, produced by reacting animal and vegetable oils or waste oils with alcohols (such as methanol or ethanol). Biodiesel has the characteristics of clean, environmentally friendly, renewable, good ignition performance, and wide applicability. Its properties are very similar to those of fossil diesel. It is a good alternative raw material and has been studied and utilized all over the world. [0003] At present, the metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08C12N9/20C12P7/64
CPCC12N9/20C12N11/08C12P7/649C12Y301/01003Y02E50/10
Inventor 王永华林森杨博王卫飞蓝东明
Owner SOUTH CHINA UNIV OF TECH
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