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Process for synthesizing ethyl caproate by yeast display lipase synthesis

A technology of ethyl hexanoate and lipase, applied in the field of preparation of ethyl hexanoate, can solve the problems of difficult separation, regeneration, recycling, high production cost, long reaction time, etc., and achieves good operational stability and reaction time. The effect of shortening and reducing production costs

Inactive Publication Date: 2008-10-15
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficulty in the separation, regeneration and recycling of enzymes, the reaction time is long, and the enzymes are easily inactivated during the production process. If a high-activity lipase such as Novo435 is used, the price is expensive, and the cost of applying it to large-scale industrial production is too high.

Method used

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  • Process for synthesizing ethyl caproate by yeast display lipase synthesis

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Experimental program
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Embodiment 1

[0018] (1) Production of Pichia pastoris whole-cell enzyme preparation displaying Candida antarctica lipase B (calb for short) on the surface.

[0019] Firstly, primers were designed according to the nucleotide sequence of Candida antarctica lipase (calb) gene LF058, and full-length calb was amplified by PCR using Candida antarctica genomic DNA as a template. 30 cycles of 94°C for 1min, 60°C for 1min, and 72°C for 1min, and 72°C for 10min. 100 μL of the PCR amplification product was digested with Eco RI and Not I, and 20 μL of the plasmid pPIC9K-Flop1 was digested with Eco RI and Not I, and purified with T 4 DNA ligase ligation, ligation with pMD18-T, transformation of E.coli Top10, and screening of positive transformants.

[0020] The verified recombinant plasmid pKFS-CALB was linearized by Sac I and transformed into P. pastorisGS115 by electrotransfer. The transformed products were spread on a G418 gradient plate and cultured at 30°C for 2 days. Pick part of the transform...

Embodiment 2

[0023] (1) Production of Pichia pastoris whole-cell enzyme preparation displaying Rhizomucor miehei lipase (abbreviated as RML).

[0024] Primers were designed according to the Rhizomucor miehei lipase RML precursor sequence (P19515, GI: 417256) reported in GenBank, and the plasmid PMD18-T-RML was used as a template for PCR amplification. The PCR system is template 1 μL; 10×Taq DNA polymerase buffer 5 μL (containing Mg2+); 2.5 mmol / L dNTP 4 μL; 20 μM mol / L upstream and downstream primers 1 μL each; Taq DNA polymerase 0.75 μL, add sterile water to the total volume 50 μL. The reaction conditions are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 45°C for 45 s, and extension at 72°C for 2 min, a total of 30 cycles; the 30th cycle was extended at 72°C for 10 min, and both the PCR product and the pKFS plasmid were treated with SnaBI and AvrII Double enzyme digestion, after constructing the recombinant plasmid pPIC9K-RML, use CaCl 2 The transforma...

Embodiment 3

[0028] (1) Production of Pichia pastoris whole-cell enzyme preparation displaying Rhizomucor miehei lipase (abbreviated as RML).

[0029] Primers were designed according to the Rhizomucor miehei lipase RML precursor sequence (P19515, GI: 417256) reported in GenBank, and the plasmid PMD18-T-RML was used as a template for PCR amplification. The PCR system is template 1 μL; 10×Taq DNA polymerase buffer 5 μL (containing Mg2+); 2.5 mmol / L dNTP 4 μL; 20 μM mol / L upstream and downstream primers 1 μL each; Taq DNA polymerase 0.75 μL, add sterile water to the total volume 50 μL. The reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 45°C for 45 s, and extension at 72°C for 2 min, a total of 30 cycles; the 30th cycle was extended at 72°C for 10 min, and both the PCR product and the pKFS plasmid were double-coated with SnaBI and AvrII. Restriction digestion, after constructing the recombinant plasmid pPIC9K-RML, use the CaCl2 transf...

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Abstract

The invention discloses a method for synthesizing ethyl caproate under the catalysis of yeast display lipase. The method comprises two steps, namely the production of full cellular zymin and the synthesis of ethyl caproate. The method comprises the following: a step of cloning the lipase gene into the Pichia yeast surface display vector pKFS to construct the Pichia yeast surface display expression vector pKFS-lipase which is transformed into the host bacteria of Pichia yeast through linearization, a yeast genetic engineering bacteria capable of displaying active pheron on the surface of the Pichia yeast being obtained through screening, and the engineering bacteria being fermented in a rocking bottle to obtain a thallus which is used to make the full cellular zymin after 24 hours of vacuum freeze drying; and then a step of adopting caproic acid and alcohol as raw material which is esterified at the action of the biocatalyst of yeast full cellular zymin with the lipase displayed on the surface to obtain the ethyl caproate product. The method is capable of collecting thalli centrifugally for reuse after the reaction with short reaction time, high yield and good operational stability, thereby greatly reducing production costs.

Description

technical field [0001] The invention relates to a preparation method of ethyl caproate, in particular to a new production process for efficiently catalyzing and synthesizing ethyl caproate with a whole cell enzyme preparation. Background technique [0002] Ethyl caproate is a commonly used flavoring substance in the brewing industry, and is the main flavor substance of Luzhou-flavor liquor (such as Wuliangye). Its content directly affects the quality of koji liquor and is also one of the important indicators of liquor quality. Every year, more than 3,000 tons of ethyl caproate are used in my country for flavoring Luzhou-flavor koji wine, worth hundreds of millions of yuan. At present, the production of ethyl caproate marked with food grade basically adopts chemical synthesis, using sulfuric acid or p-toluenesulfonic acid as a catalyst, and direct esterification reaction by caproic acid and ethanol. Ethyl caproate synthesized by chemical method will produce "floating fragran...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12N9/20C12N15/57C12N15/81C12N1/19C12R1/84
Inventor 林影韩双艳潘志友郑穗平黄登峰
Owner SOUTH CHINA UNIV OF TECH
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