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Method for preparing biodiesel from pichia pastoris whole-cell lipase catalysis

A technology of biodiesel and Pichia pastoris, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve good economic and social benefits, simple preparation process, and good short-alcohol tolerance

Active Publication Date: 2014-05-14
NANNING XINKEJIAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, the recombinant Pichia pastoris whole-cell catalyst expressed intracellularly and its biodiesel catalytic application have not been reported yet.

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  • Method for preparing biodiesel from pichia pastoris whole-cell lipase catalysis

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Effect test

Embodiment 1

[0030] The method for preparing biodiesel catalyzed by Pichia pastoris whole cell lipase of the present embodiment is:

[0031] (1) Recombination and transformation:

[0032] By constructing a recombinant plasmid of the target lipase gene, transforming the recombinant plasmid, and screening transformants containing a high-copy target gene, high-efficiency intracellular expression of recombinant yeast engineering bacteria is obtained;

[0033] The recombinant plasmid is constructed by connecting pPICZA with the target lipase gene, and then transforming Pichia pastoris competent cells to obtain transformants of the high-copy target gene; the transformants of the high-copy gene are obtained at a concentration of 1000 μg / ml 。 A transformant capable of normal growth on the bleomycin (Zeocin) resistant YPD plate; the Pichia pastoris is Pichia pastoris GS115.

[0034] (2) Induction:

[0035] Through the culture method of recombinant yeast engineered bacterial cell accumulation ...

Embodiment 2

[0050] The method for preparing biodiesel catalyzed by Pichia pastoris whole cell lipase of the present embodiment is:

[0051] (1) Recombination and transformation:

[0052] By constructing a recombinant plasmid of the target lipase gene, transforming the recombinant plasmid, and screening transformants containing a high-copy target gene, high-efficiency intracellular expression of recombinant yeast engineering bacteria is obtained;

[0053] The recombinant plasmid is constructed by connecting pPICZA with the target lipase gene to construct a recombinant plasmid, and then transforms Pichia pastoris competent cells to obtain a transformant with a high copy of the target gene; / ml. A transformant capable of normal growth on the bleomycin (Zeocin) resistant YPD plate; the Pichia pastoris is Pichia pastoris X33.

[0054] (2) Induction:

[0055] Through the culture method of recombinant yeast engineered bacterial cell accumulation stage and lipase induction expression stage, ...

Embodiment 3

[0070] The method for preparing biodiesel catalyzed by Pichia pastoris whole cell lipase of the present embodiment is:

[0071] (1) Recombination and transformation:

[0072] By constructing a recombinant plasmid of the target lipase gene, transforming the recombinant plasmid, and screening transformants containing a high-copy target gene, high-efficiency intracellular expression of recombinant yeast engineering bacteria is obtained;

[0073] The recombinant plasmid is constructed by connecting pPICZA with the target lipase gene, and then transforms Pichia pastoris competent cells to obtain transformants of high-copy target genes; ml. A transformant capable of normal growth on the bleomycin (Zeocin) resistant YPD plate; the Pichia pastoris is Pichia pastoris KM71.

[0074] (2) Induction:

[0075] Through the culture method of recombinant yeast engineered bacterial cell accumulation stage and lipase induction expression stage, high-density production of recombinant yeast c...

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Abstract

The invention discloses a method for preparing biodiesel from pichia pastoris whole-cell lipase catalysis. Recombined pichia pastoris whole-cells are prepared by taking lipase genes, a recombination carrier and a yeast host as starting materials through yeast recombination expression and high-density fermentation based on high-efficiency intracellular expression lipase; meanwhile, due to permeability treatment, permeation type yeast whole-cells are applied to biodiesel catalysis, so that the high-yield biodiesel can be prepared. A recombined pichia pastoris whole-cell catalyst disclosed by the invention is simple in preparation process, is pollution-free and is a good biological catalyst for preparing the biodiesel in a green way; the recombined pichia pastoris whole-cell catalyst can be applied to a broad-spectrum oil source and is high in short-alcohol-refining tolerance; compared with other whole-cell catalysts, the recombined pichia pastoris whole-cell catalyst can be prepared in large scale through the high-density fermentation, so that the cost is low, and the recombined pichia pastoris whole-cell catalyst has high economical and social benefits.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and energy, and relates to a method for preparing biodiesel, in particular to a method for preparing biodiesel catalyzed by whole-cell lipase of Pichia pastoris. Background technique [0002] Biodiesel is a long-chain fatty acid methyl ester or ethyl ester produced by the transesterification reaction of animal and vegetable oils and short-chain alcohol methanol or ethanol under the action of a catalyst. It is a kind of environmentally friendly, renewable, and can replace petrochemical diesel. clean energy. Catalysts for preparing biodiesel can be divided into acid-base catalysts and lipase catalysts. Acid-base catalysis has defects such as environmental pollution and corrosion of production equipment. For waste oils with high acid value (such as waste oil), alkali catalysis will also form a saponification reaction, which hinders the further progress of the reaction. Lipase catalysis can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/64C12N15/81C12R1/84
CPCY02E50/10
Inventor 阎金勇蒋永强钟志健卢汉狼吕军潘勤春甘敏鑫
Owner NANNING XINKEJIAN BIOTECH
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