Method for preparing biodiesel catalyzed by Pichia pastoris whole cell lipase

A technology of biodiesel and Pichia pastoris, which is applied to the preparation of biodiesel, and the field of Pichia pastoris whole cell lipase catalyzed to prepare biodiesel, achieves the effects of good tolerance, good economic and social benefits, and a simple preparation process

Active Publication Date: 2016-11-30
NANNING XINKEJIAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 catalyzed by Pichia pastoris whole cell lipase

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Experimental program
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Effect test

Embodiment 1

[0030] The method that the Pichia pastoris whole cell lipase of the present embodiment catalyzes the preparation of biodiesel is:

[0031] (1) Recombination and transformation:

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

[0033] The recombinant plasmid is constructed by connecting pPICZA and the target lipase gene, and then transforming Pichia pastoris competent cells to obtain a transformant of the high-copy target gene; the transformant of the high-copy gene is at a concentration of 1000 μg / ml 。 Transformants that can grow normally on bleomycin (Zeocin) resistant YPD plates; the Pichia pastoris is Pichia pastoris GS115.

[0034] (2) Induction:

[0035] Through the culture mode of recombinant yeast engineering bacteria cell accumulation...

Embodiment 2

[0050] The method that the Pichia pastoris whole cell lipase of the present embodiment catalyzes the preparation of biodiesel is:

[0051] (1) Recombination and transformation:

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

[0053] The recombinant plasmid is constructed by linking pPICZA and the target lipase gene, and then transforming Pichia pastoris competent cells to obtain a high-copy target gene transformant; the high-copy gene transformant is at a concentration of 2000 μg / ml. Transformants that can grow normally on bleomycin (Zeocin) resistant YPD plates; the Pichia pastoris is Pichia pastoris X33.

[0054] (2) Induction:

[0055] Through the culture mode of recombinant yeast engineering bacteria cell accumulation stage and lipase induc...

Embodiment 3

[0070] The method that the Pichia pastoris whole cell lipase of the present embodiment catalyzes the preparation of biodiesel is:

[0071] (1) Recombination and transformation:

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

[0073] The described recombinant plasmid is that pPICZA is connected with the target lipase gene to construct a recombinant plasmid, and then transformed into Pichia pastoris competent cells to obtain a transformant of the high-copy target gene; the transformant of the high-copy gene is at a concentration of 500 μg / ml. Transformants that can grow normally on bleomycin (Zeocin) resistant YPD plates; the Pichia pastoris is Pichia pastoris KM71.

[0074] (2) Induction:

[0075] Through the culture mode of recombinant yeast en...

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Abstract

The invention discloses a method for preparing biodiesel catalyzed by whole-cell lipase of Pichia pastoris. The method uses lipase gene, recombinant vector and yeast host as starting materials, utilizes yeast recombinant expression and high-density fermentation, and is based on high-efficiency intracellular Expressing lipase to prepare whole cells of recombinant Pichia pastoris, combined with permeabilization treatment, applying whole cells of permeable yeast to biodiesel catalysis to prepare biodiesel with high yield. The whole-cell catalyst of recombinant Pichia pastoris of the present invention has a simple preparation process and no pollution, and is an excellent biocatalyst for green biodiesel preparation; the whole-cell catalyst of recombinant Pichia pastoris can adapt to a broad-spectrum oil source and has good short refining Alcohol tolerance, compared with other whole-cell catalysts, is easier to prepare on a large scale through high-density fermentation, so the cost is low, and it has better economic 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 Pichia pastoris whole-cell lipase. Background technique [0002] Biodiesel is a long-chain fatty acid methyl ester or ethyl ester produced by transesterification of animal and vegetable oils and short-chain alcohol methanol or ethanol under the action of a catalyst. 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 oil 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 overcome the above-mentioned defects of acid-base catalysis and has attracted attention from academia and in...

Claims

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Application Information

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