Preparation method of biodiesel and compound enzyme used by preparation method

A biodiesel and compound enzyme technology, applied in the field of compound enzymes, can solve problems such as industrial application limitations and high prices, and achieve the effects of overcoming substrate specificity, easy separation, and environmental friendliness

Inactive Publication Date: 2013-02-06
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, Novozym’s Novozym 435 (CALB) is currently the most widely used commercial immobilized lipase in biodiesel production. Due to its high price, 2270 US dollars per kilogram, its industrial application is limited.

Method used

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  • Preparation method of biodiesel and compound enzyme used by preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the cloning of CALB gene,

[0027] Primers: Upstream primer: 5'-CCCGAATTCGCCACTCCTTTGGTGAAGC-3' (contains EcoRI restriction site); downstream primer: 5'-CCCGGTACCTCAGGGGGTGACGATGCCGGAGCAG-3' (contains KpnI restriction site).

[0028] PCR reaction system: 1 μL Candida antarctica (NRRL No.Y-7954) genomic DNA, 1 μL upstream primer, 1 μL downstream primer, 25 μL Premix ExTaq, 22 μL ultrapure water.

[0029] PCR reaction conditions: Denaturation at 94°C for 10 min; denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min; incubation at 4°C.

[0030] The yield and specificity of the PCR products were detected by 1% agarose gel electrophoresis, and purified with a PCR product recovery kit (BIOMIGA, Shanghai).

[0031] The purified PCR amplification product was ligated with the cloning vector pMD-19T, and the ligated liquid was electrotransformed into E. coli-TOP10 competent cells, and a s...

Embodiment 2

[0032] Embodiment 2: the cloning of ROL gene,

[0033] Primers: Upstream primer: 5'-CCCGAATTCATGGTTCCTGTTTCTGGTAAATC-3' (contains EcoR I restriction site); downstream primer: 5'-CCCTCTAGATTACAAACAGCTTCCTTCGT-3' (contains Xba I restriction site), PCR reaction system: 1 μL R. oryzae (DSM 853) genomic DNA, 1 μL upstream primer, 1 μL downstream primer, 25 μL Premix ExTaq, 22 μL ultrapure water.

[0034] PCR reaction conditions: Denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, annealing at 52°C for 1 min, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min; incubation at 4°C.

[0035] The yield and specificity of the PCR products were detected by 1% agarose gel electrophoresis, and purified with a PCR product recovery kit (BIOMIGA, Shanghai).

[0036] The purified PCR amplification product was ligated with the cloning vector pMD-18T, and the ligated liquid was electrotransformed into E. coli-TOP10 (NOVAGEN) competent cells, and a small amount of pl...

Embodiment 3

[0037] Example 3: Construction of Pichia pastoris expression vector.

[0038]Use the designed primers with restriction sites (same as in Examples 1 and 2), and use the constructed pMD19T-CALB (Example 1) and pMD18T-ROL (Example 2) recombinant plasmids as templates to perform PCR Amplify (the reaction system and conditions are the same as in Examples 1 and 2), detect by gel electrophoresis, and recover the PCR amplification product by tapping the gel. The purified sample of the PCR product of the target gene and the vector pPICZαA (purchased from Invitrogen) were subjected to double enzyme digestion (the restriction sites used by CALB are EcoR I and Kpn I, and the restriction sites used by ROL are EcoR I and Xba I).

[0039] The digested PCR products were resuspended with the pPICZαA vector after concentration and added to 8 μL of sterile water, 1 μL of 10× Ligase Buffer and 1 μL of Ligase were added, and ligated overnight at 16°C. Transfer the ligated solution into E. coli-TO...

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Abstract

The invention discloses a preparation method of biodiesel and compound enzyme used by the method. The method comprises the following steps of: adding short-chain alcohol, oil, water and the compound enzyme into a reaction vessel, controlling the temperature at 30-50 DEG C, and reacting for 48-72hours to prepare the biodiesel, wherein the molar ratio of the short-chain alcohol to the oil is (3-5): 1, the use level of the water is 40-100% as heavy as that of the oil, and the use level of the compound enzyme is 4-22U/g of soybean oil. The method is environment-friendly without adding any organic solvents, can express the compound enzyme by utilizing recombinant bacteria, does not need to purchase the commercial enzyme, and the compound lipase is adopted to cooperatively catalyze, so that the substrate specificity of single lipase can be overcome, the ester converting efficiency when the biodiesel is prepared by an enzyme method can be improved, and products are easy to separate. After the method for catalysis by using the compound lipase is used, the use level of the required enzyme is reused by more than 50% of the use level of the single lipase, and the yield of the active ingredients in the biodiesel, i.e., the fatty acid methyl ester, is 98%, so that the production cost can be greatly reduced. The preparation method is very good in industrialized prospect.

Description

technical field [0001] The invention belongs to the technical field of preparing biodiesel by a biological enzymatic method, and in particular relates to a method for preparing biodiesel by composite synergistic catalysis of lipases from two different sources and a composite enzyme used therefor. Background technique [0002] Fossil energy is the material basis for the survival and development of modern society. With the rapid development of the global economy, the world's energy consumption is also increasing, which makes people realize that it is an urgent matter to alleviate the energy crisis and seek alternatives to fossil energy. Biodiesel is just such a kind of renewable and clean fuel prepared from animal and vegetable oils and fats through transesterification process, which belongs to one of biomass energy. Compared with traditional petrochemical diesel, biodiesel has good fuel performance, high safety performance, low-temperature starting performance of the engine,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C10G3/00C11C3/10C10L1/02C12P7/64
CPCY02E50/13Y02E50/10Y02P30/20
Inventor 李迅邓若冰王飞李治林时号
Owner NANJING FORESTRY UNIV
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