Pichia pastoris engineering bacteria of surface display candida antarctica lipase B (CALB) and method for catalyzing and synthesizing short-chain aromatic ester

A Pichia pastoris, surface display technology, which is applied in the field of whole-cell enzyme preparations to efficiently catalyze the synthesis of short-chain aromatic esters, can solve the problems of high cost, long production cycle, shortened reaction cycle, etc., and achieves short reaction time and reduced production cost. , the effect of high yield

Active Publication Date: 2010-08-04
SOUTH CHINA UNIV OF TECH
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AI-Extracted Technical Summary

Problems solved by technology

[0006] Chinese invention patent CN1223300 discloses a method of microbial lipase enzymatic synthesis of ester, the enzyme used is the self-produced Rhizopus sinica lipase or commercial immobilized enzyme Lypozyme IM lipase, the catalytic reaction tim...
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Method used

[0036] Reagents are fully dehydrated with molecular sieves in advance. Add isoamyl alcohol and butyric acid to n-heptane. After the addition, the concentration of isoamyl alcohol was 0.88 mol/L, and the concentration of butyric acid was 0.8 mol/L. Take 10mL of substrate (734.3 μL of butyric acid, 957.7 μL of isoamyl alcohol, 8308 μL of n-heptane, and the acid-alcohol molar ratio is 1:1.1) mixture in a 50 mL Erlenmeyer flask with a stopper, and add 20 g/L of Example 1 Prepared thalli...
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Abstract

The invention discloses Pichia pastoris engineering bacteria of surface display candida antarctica lipase B (CALB) and a method for catalyzing and synthesizing short-chain aromatic ester. The method comprises the following steps of: fermenting a bacterial strain shake flask of the Pichia pastoris engineering bacteria of the surface display CALB to gain thalli; freezing and drying in vacuum for above 24h to prepare a whole-cell enzyme preparation; carrying out esterification reaction to obtain a short-chain aromatic ester product by adopting short-chain acid and short-chain alcohol as raw materials, the whole-cell enzyme preparation as a catalyst and liquid which can dissolve the short-chain acid and the short-chain alcohol as a solvent, wherein the temperature of the esterification reaction is 20-60 DEG C, the consistency of the whole-cell enzyme preparation of a reactant in a reaction system is 10-40g/L, and the reaction time is 1-6h. Compared with the prior report, the method greatly shortens the reaction time, has a productive rate as high as 98 percent and high continuous operating stability, can recycle the whole-cell catalyst which is centrifugally recovered after the reaction and greatly reduce production cost.

Application Domain

Technology Topic

Examples

  • Experimental program(13)

Example Embodiment

[0025] Example 1
[0026] Production of Pichia whole-cell enzyme preparation displaying Candida antarctica lipase B on the surface.
[0027] First, the primers were designed according to the nucleotide sequence of the Candida antarctic lipase (calb) gene LF058 (upstream primer is 5'TGTAGAATTCCTGCCTTCCGGTTCGGACCCTG 3', and EcoR I restriction site is introduced, and the downstream primer 5'GAGGCCGTAGCAGTGGGGATGCGCATAGAGCTCAGGTCCTCCACGAG 3', introduced Mlu I restriction site). Using Candida antarctica genomic DNA as a template for PCR amplification of full-length calb, the PCR procedure is as follows: 95°C pre-denaturation for 5 minutes, 94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute for 30 cycles, and 72°C for 10 minutes of extension.
[0028] Secondly, design primers according to the nucleotide sequence of α lectin. The upstream primer is 5'CTCCGGCATCGTCACCCCTACGCGTATCTCGAGTCCAGGAGGTGCTC 3', which introduces the 19bp and MluI restriction site at the downstream end of CALB gene, and the downstream primer is 5'ATTAGCGGCCGCTTAGAATAGCAGGTACGACAAAAG 3', which introduces Not I restriction enzyme digestion. Site. Using the recombinant plasmid containing α lectin as a template for PCR amplification of α lectin. The PCR program is as follows: pre-denaturation at 94°C for 5 min, 30 cycles at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, and extension at 72°C for 10 min.
[0029] 1μL of the above two PCR products were taken as a template and added to the new PCR system to amplify the CALB-NS fusion gene fragment (upstream primer used to amplify the upstream primer of CALB: 5'TGTAGAATTCCTGCCTTCCGGTTCGGACCCTG 3', downstream primer used to amplify the downstream of α lectin Primer: 5'ATTAGCGGCCGCTTAGAATAGCAGGTACGACAAAAG 3'). The PCR program is as follows: pre-denaturation at 94°C for 5 min, 30 cycles at 95°C for 1 min, 55°C for 1.5 min, and 72°C for 2 min, and extension at 72°C for 10 min. The fusion gene product was purified and digested with Eco RI and Not I. At the same time, 20 μL plasmid pPIC9K was digested with Eco RI and Not I. After purification, it was digested with T 4 DNA ligase ligation, transformation of E. coli Top10, selection of positive transformants.
[0030] The verified recombinant plasmid pKNS-CALB was linearized by Sac I and transformed into P.pastoris GS115 by electrotransfer. The transformant was coated on the G418 gradient plate and incubated at 30°C for 2 days. Part of the transformants resistant to high concentration of G418 were selected and inoculated into 5ml YPD culture medium, incubated overnight at 30°C and 250r/min, and the lipase activity was detected by alkaline titration to obtain a Pichia pastoris engineered strain (Pichia pastoris GS115/ pKNS-CALB), this strain was deposited in the China Type Culture Collection on September 30, 2009, the deposit number is: CCTCC NO: M 209217, and the deposit address is Wuhan University, Wuhan City, Hubei Province (Zip code 430072). The engineering strain of Pichia pastoris was first cultivated with YPD seed solution and then expanded by BMGY for 24 hours; the bacteria were collected by centrifugation at 6000g at room temperature. Then use the same volume of BMMY to induce culture, and add methanol every 24h to a final concentration of 1%. Incubate on a shaker at 30°C and 250 rpm for 4 days. Centrifuge at 7000 rpm and 4°C for 10 minutes, discard the supernatant, wash the cells three times with deionized water, and then resuspend the cells and freeze-dry them in a vacuum for 24 hours to obtain freeze-dried cells.

Example Embodiment

[0031] Example 2
[0032] Using acetic acid and ethanol as substrates, whole-cell enzyme preparations catalyze the non-aqueous phase esterification reaction to prepare ethyl acetate.
[0033] Reagents are used in advance The molecular sieve fully removes water. Add absolute ethanol and glacial acetic acid to n-heptane. After the addition, the concentration of absolute ethanol is 0.5 mol/L, and the concentration of glacial acetic acid is 0.4 mol/L. Take 10mL of the substrate (including 288.6μL of glacial acetic acid, 291.2μL of absolute ethanol, 9420.2μL of n-heptane, and the molar ratio of acid to alcohol is 1:1.25) and place the mixture in a 50mL Erlenmeyer flask with stopper, and add the content of 30g/L to the example 1. The prepared lyophilized bacterial powder, the reaction temperature is 40℃, and the reaction is shaken at 200rpm. After 0.5h, 0.6g molecular sieve is added and reacted for 3h. The conversion rate of acetic acid can reach 92.1%. The reaction time is only a report that the yield is close. 1/26 of that.

Example Embodiment

[0034] Example 3
[0035] Using butyric acid and isoamyl alcohol as substrates, whole-cell enzyme preparation catalyzes the non-aqueous phase esterification reaction to prepare isoamyl butyrate.
[0036] Reagents are used in advance The molecular sieve fully removes water. Add isoamyl alcohol and butyric acid to n-heptane. After the addition, the concentration of isoamyl alcohol is 0.88 mol/L, and the concentration of butyric acid is 0.8 mol/L. Take 10 mL of the substrate (of which 734.3 μL of butyric acid, 957.7 μL of isoamyl alcohol, 8308 μL of n-heptane, and the molar ratio of acid to alcohol is 1:1.1) and place the mixture in a 50 mL Erlenmeyer flask with stopper, and add the content of Example 1 with a content of 20 g/L The prepared freeze-dried bacterial powder, the reaction temperature is 50 ℃, and then the reaction is shaken at 200 rpm. After 0.5 h, 0.6 g molecular sieve is added, and the reaction is 3 h. The conversion rate of butyric acid can reach 97%, as reported by Yang Benhong (Yang Benhong. Non-aqueous Phase lipase-catalyzed synthesis of isoamyl butyrate. Compared with the lipase-catalyzed synthesis of isoamyl butyrate produced by Rhizopus PW358 of China Food Additives, 2005, 5:74-79) through solid-state fermentation, the reaction time is shortened by 2h .
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Description & Claims & Application Information

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