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A method for synthesizing short-chain aromatic esters using cutinase

A technology of cutinase and aromatic ester, applied in the fields of enzyme engineering and fermentation engineering, can solve the problems of low stability, hindered application, increased concentration, etc., and achieves the effects of improving content, good activity and stability, and simple reaction method

Active Publication Date: 2019-03-15
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most lipases generally have better specific selectivity for long-chain substrates, and their relatively low stability hinders their application in large-scale industrial production.
In addition, during the fermentation process of liquor grains / rice wine, the concentration of ethanol and acid will continue to rise, and it is difficult for exogenously added enzymes to play the expected role in the complex liquor fermentation environment

Method used

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  • A method for synthesizing short-chain aromatic esters using cutinase
  • A method for synthesizing short-chain aromatic esters using cutinase
  • A method for synthesizing short-chain aromatic esters using cutinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction and shake flask fermentation of P. pastoris KM71 / pPIC9K-ScCuA

[0032](1) According to the cutinase gene published by NCBI accession number: KF193402, the recombinant plasmid pPIC9K-ScCuA was obtained by chemical synthesis; after linearization by SacI endonuclease, it was electrotransformed into P. pastoris KM71, and the transformation mixture was spread on the MD plate , cultured at 30°C for 2-3d, picked a single colony and planted it on the YPD / G418 plate, the G418 concentration screening gradient was 0mg / mL, 1mg / mL, 1.5mg / mL, 2mg / mL, cultured at 30°C for 1-2d ;Pick the single clone on the YPD / G418 concentration gradient plate in a 50mL Erlenmeyer flask filled with 10mL of YPD medium, culture at 30°C and 200r / min for 20-24h, draw 500μL of the bacterial liquid into a glycerol tube, and store at -80°C refrigerator.

[0033] (2) Pick the single clones on the YPD / G418 concentration gradient plate in 10mL YPD medium, culture in a 200r / min constant ...

Embodiment 2

[0035] Example 2: Fermentation process of P.pastoris KM71 / pPIC9K-ScCuA

[0036] 1. Strain P. pastoris KM71 / pPIC9K-ScCuA.

[0037] 2. Seed culture: Take out the glycerol tube stored in the refrigerator at -80°C, inoculate 200 μL of bacterial solution into 100 mL of seed medium, and cultivate it in a shaker at 30°C and 200 rpm for about 24 hours; the composition of the seed medium is: 10 g of yeast powder / L, peptone 20g / L, YNB 13.4g / L, glycerin 30g / L, biotin 4×10 -4 g / L.

[0038] 3. The amount of fermentation inoculum is 10%.

[0039] 4. Fermentation medium (BSM) composition: CaSO 4 .2H 2 O 0.939g / L, MgSO 4 ·7H 2 O 14.9g / L, KOH4.13g / L, K 2 SO 4 18.2g / L, glycerol 30.0g / L, 85%H 3 PO 4 26.7mL / L, trace element solution PTM 1 4.3mL / L; trace element solution PTM 1 Composition: FeSO 4 ·7H 2 O 65g / L, KI 0.08g / L, H 2 SO 4 5.0g / L, MnSO 4 ·H 2 O 3g / L, CuSO 4 ·5H 2 O 6g / L, H 3 BO 3 0.02g / L, Na 2 MoO 3 2H 2 O 0.2g / L, CoCl 2 0.5g / L, ZnCl 2 20g / L; feed growth medium...

Embodiment 3

[0043] Example 3: Purification and Enzymatic Properties of Recombinant Cutinase

[0044] Add 35% solid ammonium sulfate to the fermentation supernatant for salting out overnight, centrifuge at 8000rpm for 20min at 4°C, dissolve the precipitate with an appropriate amount of 1×Mcilvation buffer with pH 7.0, use a dialysis membrane to remove residual ammonium sulfate, and dialyze the sample The loaded sample was made after filtering through a 0.4um membrane. After DEAE-Sepharose anion exchange resin. The SDS-PAGE electrophoresis of the protein during purification is shown in figure 1 .

[0045] 1. Purification of recombinant cutinase:

[0046] After purification, the specific activity of the recombinant cutinase increased from 90.6U / mg to 149.1U / mg, the purification factor was 1.6 times, and the recovery rate was 31.1%.

[0047] 2. Enzymatic properties include optimum pH, optimum temperature, pH stability, temperature stability and stability in organic solvents of the recombi...

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Abstract

The invention relates to a method for synthesizing a short-chain flavor ester with cutinase and belongs to the technical field of enzyme engineering and fermentation engineering. According to the method, pichia pastoris serves as a host bacterium to build a genetically engineered bacterium of overexpressed cutinase, the genetically engineered bacterium is induced to generate cutinase through fermentation, and enzyme activity of cutinase in supernatant of fermentation liquor reaches 420 U / mL. The recombinant enzyme is further used to produce the short-chain flavor ester in an organic phase, Chinese rice wine aging acceleration and baijiu solid fermentation. The method has the advantages that an enzyme source preparation method is simple, cost is low, enzyme quantity is large, a short-chain flavor ester production method is simple, the conversion rate is high, the yield is high and time is short, and industrial enlarged production is facilitated.

Description

technical field [0001] The invention relates to a method for synthesizing short-chain aromatic esters by using cutinase, which belongs to the technical fields of enzyme engineering and fermentation engineering. Background technique [0002] Short-chain aromatic esters are composed of short-chain fatty acids (C 2 -C 10 ) and alcohol (C 2 -C 6 ) synthetic esters, which account for a large proportion in the composition of natural spices, are widely used in food, cosmetics, beverages and other industries. For example, ethyl acetate is widely used in the production of fruit flavors and various spices, and its total consumption level in the world reached 2800kt / a in 2014. Ethyl butyrate has a pineapple-like aroma and is widely used in the preparation of various food, wine and tobacco flavors. As the main aromatic substance of Luzhou-flavor liquor, the national output of ethyl caproate has exceeded 3000 tons. Another example is ethyl lactate with wine aroma, its special aroma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/64C12P7/62C12H1/15C12G3/02C12N15/81C12N9/18C12R1/84
CPCC12G3/02C12H1/003C12N9/18C12P7/62C12P7/6436C12Y301/01074
Inventor 吴敬宿玲恰郭小杰
Owner JIANGNAN UNIV
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