Lipase mutant with high esterification activity

A technology of fatty acid esters, animal and vegetable oils, applied in the direction of enzymes, hydrolytic enzymes, biochemical equipment and methods, etc., can solve problems such as difficult recycling, complicated process, and easy deterioration

Active Publication Date: 2018-07-03
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] However, chemical synthesis of biodiesel has the following disadvantages: the process is complicated, the alcohol must be excessive, and the subsequent process must have a corresponding alcohol recovery device, which requires high energy consumption; the color is deep, because the unsaturated fatty acids in the fat are easy to deteriorate at high temperatures; the esterification product is difficult to obtain. Recycling, high cost; waste lye...

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  • Lipase mutant with high esterification activity
  • Lipase mutant with high esterification activity
  • Lipase mutant with high esterification activity

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preparation example Construction

[0105] The polypeptide of the invention has high esterification activity and can be used to prepare biodiesel. Therefore, the present invention provides a method for preparing biodiesel, said method comprising the step of using the polypeptide of the present invention to synthesize biodiesel.

[0106] Biodiesel refers to fatty acid monoalkyl esters obtained by transesterification of animal and vegetable oils (fatty acid triglycerides) with organic alcohols (such as methanol or ethanol), the most typical being fatty acid methyl esters. In the present invention, animal and vegetable oils can be animal and vegetable oils known in the art to produce biodiesel, including but not limited to crude oleic acid, acidified oil, a mixture of PFAD and fatty acid esters, and waste catering oil or one of them or a combination of several.

[0107] Therefore, the preparation method of the biodiesel of the present invention includes the step of using the polypeptide of the present invention to c...

Embodiment 1

[0152] Embodiment 1: TDL and TDLm expression and performance detection

[0153]The amino acid sequence of TDL is derived from the amino acid sequence of wild-type Thermomyces dupontii lipase, which is derived from NCBI, GenBank Accession: AEE61324.1. The 1-22 position of Thermomyces dupontii lipase is a signal peptide, and the 23-291 position is a mature protein. The mature protein at position 23-291 of Thermomyces dupontii lipase was selected for expression, the DNA sequence SEQ ID NO:1 was designed according to the codon preference of Pichia pastoris, and the leader peptide sequence of α-factor was added (from the DNA sequence of the commercial vector pPIC9K ), sent to Shanghai Sangon Bioengineering Co., Ltd. for whole gene synthesis, and cloned the DNA sequence into the pAO815 plasmid to obtain the pAO-TDL expression vector. The 231th Thr of the mature peptide was mutated to Arg, the 232nd Lys was mutated to Arg, and the 233rd Asn was mutated to Arg to obtain the DNA seque...

Embodiment 2

[0162] Example 2: TDLm-1 mutant library construction, screening and performance testing

[0163] 1. Mutant library construction and mutant screening

[0164] Using the pAO-TDLm vector as a template, use Taq enzyme (TaKaRa) and primer pair EPTDL-1 / EPTDL-2:

[0165] EPTDL-1: 5'-ccgGAATTCCGAAACGATGAGATTTCCTTC-3' (SEQ ID NO:8)

[0166] EPTDL-2: ccgGAATTCTCACAAACAAGTGCC (SEQ ID NO:9)

[0167] Perform error-prone PCR (additional 0.3mM MnCl during PCR 2 ) to obtain a collection of mutant amplicon fragments with a size of about 1000 bp. The obtained fragment was cloned into pAO-TDLm through the EcoRI restriction site, and the obtained vector was transformed into Escherichia coli DH5α strain, and a total of 1×10 4 A TDLm mutant.

[0168] Will each 1×10 3 Each TDLm mutant was washed with 2 ml of sterile water into 8 ml of LB liquid medium (containing 100 μg / ml ampicillin), and cultured at 37° C. for 4 h. The plasmid was extracted, linearized with SalI, and a fragment of about 8.5...

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Abstract

The invention provides a lipase mutant with high esterification activity and particularly provides a polypeptide. The polypeptide is selected from (1) a polypeptide with an amino acid sequence represented by SEQ ID NO:7 and (2) a polypeptide formed by the sequence represented by SEQ ID NO:7 and a sequence for promoting the expression and purification of the sequence represented by SEQ ID NO:7. Theinvention further provides a polynucleotide sequence encoding the polypeptide, a corresponding nucleic acid construct, a host cell and application of the polypeptide, the polynucleotide sequence, thenucleic acid construct and the host cell in the preparation of biodiesel.

Description

technical field [0001] The invention relates to a lipase mutant with high esterification activity. Background technique [0002] Diesel, as an important petroleum refining product, occupies a relatively high share in the fuel structure of various countries and has become an important power fuel. With the acceleration of dieselization of vehicles worldwide, the demand for diesel will increase in the future, and the depletion of oil resources and the improvement of people's awareness of environmental protection have greatly promoted the pace of development of diesel alternative fuels in countries around the world, especially In the 1990s, biodiesel has attracted the attention of various countries due to its superior environmental performance. At present, the annual production of new cars in the world is about 50 million, and the number of cars in the world is about 750 million (including motorcycles). With the rapid development of the automobile industry, the consumption of ...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/63C12P7/64
CPCC12N9/20C12P7/649C12Y301/01003Y02E50/10
Inventor 宣姚吉周美凤戴小军徐正军牛其文
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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