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Recombinant Bacillus subtilis for expressing L.mesenteroides sourced sucrose phosphorylase

A technology of Bacillus subtilis and sucrose phosphorylase, which is applied in the fields of genetic engineering and enzyme engineering, and can solve the problems of incompatibility and the easy production of endotoxin by Escherichia coli

Active Publication Date: 2017-10-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the sucrose phosphorylase derived from L. mesenteroides can only be heterologously expressed in Escherichia coli, but Escherichia coli is prone to produce endotoxin, so it is not suitable for use in pharmaceuticals, cosmetics and other industries

Method used

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  • Recombinant Bacillus subtilis for expressing L.mesenteroides sourced sucrose phosphorylase
  • Recombinant Bacillus subtilis for expressing L.mesenteroides sourced sucrose phosphorylase
  • Recombinant Bacillus subtilis for expressing L.mesenteroides sourced sucrose phosphorylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Cloning of the Leuconostoc enteroconostoc sucrose phosphorylase-encoding gene and construction of an expression vector

[0041] according to figure 1 According to the process of constructing the expression vector, primers F and R were designed according to the synthetic sucrose phosphorylase gene:

[0042] F: 5'-GCGAAGCTTAAGGAGGATATTATGGAAATTCAGAACAAGGC-3'

[0043] R: 5'-GCGGGATCCTTAATTCTGGGTCAGATTATCGC-3'

[0044]The restriction sites are indicated by underlined letters. The PCR system is: 20 μM primers F and R 0.5 μL each, dNTPMix 4 μL, 5×PS Buffer 10 μL, 2.5 U / μL PrimeStar polymerase 0.5 uL, template 0.5 μL, add double distilled water to make up 50 μL. PCR conditions: pre-denaturation at 94°C for 4min; denaturation at 98°C for 10s, annealing at 55°C for 10s, extension at 72°C for 1min and 50s, 30 cycles. The PCR product was gel-recovered, purified by adding A, and then ligated with the cloning vector pMD-18T. The target gene was recovered by enzyme dig...

Embodiment 2

[0045] Embodiment 2: Transformation of recombinant plasmid pBSMuL3-sp

[0046] 1) Fresh LB plate (LB solid medium: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, 0.2g / L agar powder) pick a single colony of Bacillus subtilis (preservation number is CCTCC M 2016536) Inoculate in 5ml LB liquid medium, culture at 37°C, 200rpm for 10h.

[0047] 2) Take 2.5 mL and transfer it into 40 mL LB medium with 0.5 M sorbitol, culture at 37° C. with shaking at 200 rpm for four hours.

[0048] 3) Take all the bacteria liquid and bathe in ice water for 10 minutes, then centrifuge at 5000 rpm and 4°C for 5 minutes to collect the bacteria.

[0049] 4) Wash the cells with 40ml of pre-cooled electroporation buffer (sorbitol 0.5M, mannitol 0.5M, glucose 10%), centrifuge at 5000rpm, 4°C for 5min to remove the supernatant, and rinse 4 times in this way.

[0050] 5) Resuspend the washed bacteria in 1 mL of electroporation medium, aliquot them into 1.5 mL EP tubes, and fill each tube with 300 μl of co...

Embodiment 3

[0053] Embodiment 3: shake flask fermentation to produce enzyme

[0054] The recombinant Bacillus subtilis strain obtained in Example 2 was inoculated in LB medium, and after culturing at 37°C for 8 hours, it was transferred to TB fermentation medium with 5% inoculation amount, and then cultured at 37°C and 200 rpm for 2 hours. , and then moved to 33 ℃ constant temperature culture for 48h to produce enzymes. After the fermentation, the supernatant collected by centrifugation is the crude enzyme liquid.

[0055] LB medium (g / L): peptone 10, yeast extract 5, NaCl 10.

[0056] TB medium (g / L): peptone 10, yeast powder 24, glycerol 5, K 2 HPO 4 ·3H 2 O 16.43, KH 2 PO 4 2.31.

[0057] The enzyme activity in the crude enzyme solution was measured, and the result showed that the enzyme activity was 136U / mL. The results of protein electrophoresis showed that there was a band consistent with the theoretical molecular weight at 53kDa ( Figure 4 ).

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Abstract

The invention discloses recombinant Bacillus subtilis for expressing L.mesenteroides sourced sucrose phosphorylase, belonging to the technical fields of genetic engineering and enzyme engineering. The recombinant Bacillus subtilis is prepared by the steps of acquiring a target gene of the L.mesenteroides sourced sucrose phosphorylase by virtue of an artificially synthesized SPase gene, a design primer and PCR, constructing a recombinant plasmid pBSMuL3-SP, and converting the recombinant plasmid pBSMuL3-SP into Bacillus subtilis (CCTCC M2016536), so as to obtain the recombinant Bacillus subtilis. The recombinant Bacillus subtilis is used as a strain for fermenting and producing SPase. The recombinant Bacillus subtilis has relatively good effect when being used for preparing alpha-arbutin. By recombining and expressing the sucrose phosphorylase by taking food-safety Bacillus subtilis as an expression host, the enzyme producing level is high, the separation and purification are convenient, the fermentation raw materials are wide in sources, and the production cost is relatively low.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis expressing sucrose phosphorylase derived from L. mesenteroides, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Sucrose phosphorylase (EC 2.4.1.7, Sucrose Phosphorylase, SPase), a member of the GH13 family, is a specific enzyme that catalyzes the transfer of glucosidic bonds. It mainly catalyzes two types of reactions: one is to transfer the glucose group in 1-phosphate glucose to the receptor; the other is to transfer the glucose group in sucrose to the receptor, and the receptor includes inorganic phosphoric acid, water, phenolic hydroxyl group, alcohol Substances with hydroxyl and carboxyl groups. When the phenolic hydroxyl group is used as the acceptor, the conversion product is α-arbutin. α-Arbutin, as a high-efficiency pharmaceutical and cosmetic additive that is being promoted internationally, has a huge demand at home and ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/75C12P19/44C12R1/125
CPCC12N9/1051C12P19/44C12Y204/01007
Inventor 吴敬吴丹朱洁王淼淼宿玲恰
Owner JIANGNAN UNIV
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