Recombinant escherichia coli containing alpha-glucosidase gene and application of recombinant escherichia coli

A technology of recombinant Escherichia coli and glucosidase, applied in the direction of glycosylase, microbial-based methods, enzymes, etc., can solve the problems of thermal instability, poor solubility, disappearance of fragrance and cooling effect, and reduce production costs , high conversion rate, high conversion rate effect

Active Publication Date: 2017-11-28
深圳杉海创新技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In practical applications, L-menthol has some disadvantages, including instability when exposed to heat; its fragrance and cooling eff

Method used

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  • Recombinant escherichia coli containing alpha-glucosidase gene and application of recombinant escherichia coli
  • Recombinant escherichia coli containing alpha-glucosidase gene and application of recombinant escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0024] The screening and verification of embodiment 1 Xanthomonas campestris

[0025] Take 10g of diseased and rotten wild rapeseed leaves, directly add it to 50mL of normal saline, place it on a shaker for 30min, dilute the suspension with a gradient of normal saline, and spread it on a semi-selective NSCA medium (starch 15g / L , nutrient agar powder 23g / L, cycloheximide 100mg / L, solvent is distilled water, pH value is natural) plate, 28 ℃ aerobic culture for 48h. Pick a single colony with light yellow convex mucus and repeatedly streak and separate it on the NSCA plate, and repeat three times to obtain the purified strain IFE008 for the next step of identification.

[0026] Morphological observation:

[0027]1) Observation of colony characteristics: observe directly with the naked eye, and select colonies with a diameter between 2 and 4 mm, smooth surface, light yellow convex mucus, etc., which conform to the morphological characteristics of Xanthomonas campestris colonies. ...

Embodiment 2

[0029] Embodiment 2, preparation L-menthol-alpha-glucoside

[0030] 1. Construction of Escherichia coli that efficiently synthesizes α-glucosidase

[0031] Genomic DNA of Xanthomonas campestris (Xanthomonas campestris) CGMCC No.13990 in the mid-logarithmic growth phase was extracted using a bacterial genomic DNA extraction kit, and used as a template to perform PCR amplification with the following primers:

[0032] agl-F:5'-G GAATTC ATGTCGCAGACACCATGGTG-3' (the chemical line part is EcoR I recognition site);

[0033] agl-R:5'-CCC AAGCTT TCAGCCACGACCGACAGCAGC-3' (the underlined part is the Hind III recognition site).

[0034] The high-efficiency fidelity enzyme Primerstart of TaKaRa Company was used for PCR amplification. The PCR amplification program was: 95°C for 3 minutes; 98°C for 10s, 55°C for 15s, 72°C for 1min, 30 cycles; 72°C for 10min.

[0035] The resulting PCR product was purified using a PCR product recovery kit, connected to the pGEM-T Easy vector, constructe...

Embodiment 3

[0040] Application of embodiment 3 catalyst in the production of L-menthol-α-glucoside

[0041] 1. Catalyst activity detection

[0042] 0.5 g of wet thalline cells prepared by the method of Example 2 were resuspended in 10 mL of pH8.0 boric acid buffer (10 mmol / L H 3 BO 3 -KCl buffer solution); add the L-menthol of final concentration 5g / L and the maltose of 40g / L, under the condition of 40 ℃, 150rpm, water-bath shaker catalyzes 30min and 2h, and reaction solution is used for HPLC analysis.

[0043] 30min substrate conversion rate analysis: L-menthol was added with 5g / L, and after 30min conversion reaction, HPLC analysis showed that the remaining substrate was L-menthol with a concentration of 1.0g / L, and the formed product L-menthol - The concentration of α-glucoside is 8.2g / L, and the substrate conversion rate is above 80%.

[0044] 2h substrate conversion rate analysis: L-menthol was added 5g / L, after 2h conversion reaction, HPLC analysis showed that the remaining substr...

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Abstract

The invention discloses recombinant escherichia coli containing an alpha-glucosidase gene and application of the recombinant escherichia coli. The recombinant escherichia coli is obtained by transferring the alpha-glucosidase gene as shown in SEQ ID NO.1 into an escherichia coli host cell. The recombinant escherichia coli containing the alpha-glucosidase gene for producing L-menthol-alpha-glucoside can efficiently synthesize alpha-glucosidase in the cell; L-menthol is taken as a substrate, and maltose is taken as an auxiliary substrate to efficiently catalyze glycosylation reaction of L-menthol, the reaction is performed for 10-24 hours to obtain L-menthol-alpha-glucoside converted mash with concentration greater than 10%, wherein average production strength is greater than 5g.L<-1>.h<-1>, and a substrate conversion rate is greater than 95%; and the L-menthol-alpha-glucoside product is high in concentration, is high in conversion rate, and is beneficial for recycling and purifying L-menthol-alpha-glucoside.

Description

(1) Technical field [0001] The invention relates to a recombinant escherichia coli containing α-glucosidase gene and its application for producing L-menthol-α-glucoside. (2) Background technology [0002] L-menthol is a colorless transparent needle-like crystal; the relative molecular mass is 156.4, the melting point is 44°C, and the boiling point is 216.4°C; it is slightly soluble in water, soluble in conventional organic solvents such as cyclohexane, ethanol and benzene, and its structural formula is as follows: figure 1 shown. L-menthol has a fresh, light, diffuse smell and unique fragrance, and is widely used in cigarettes, cosmetics, toothpaste, mouthwash, chewing gum, sweets, etc. In addition, L-menthol can stimulate the cold receptors on the skin without causing actual temperature changes, exert local itching, pain relief, blood vessel relaxation, slight local anesthesia and promote drug penetration, so it can also be used for drug rubbing and topical anesthesia etc...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/46C12R1/19
CPCC12N9/2408C12P19/46C12Y302/0102
Inventor 朱林江陈小龙范永仙陆跃乐陈路易
Owner 深圳杉海创新技术有限公司
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