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A recombinant Lactococcus lactis capable of secreting laccase and its construction method and application

A technology of Lactococcus lactis and laccase, which is applied in the field of genetic engineering of enzymes, can solve the problems of low copy number, low expression amount, and difficulty in efficient gene secretion and expression in a lactic acid bacteria expression system, and achieves good thermal stability and acid and alkali resistance. , the effect of increasing the yield

Active Publication Date: 2022-06-21
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although a variety of genes have been expressed heterologously in food-grade lactic acid bacteria, due to the shortcomings of the lactic acid bacteria expression system, such as low copy number, low expression level, and difficulty in exocrine expression, it is particularly important for genes to be efficiently secreted and expressed in lactic acid bacteria. difficulty

Method used

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  • A recombinant Lactococcus lactis capable of secreting laccase and its construction method and application
  • A recombinant Lactococcus lactis capable of secreting laccase and its construction method and application
  • A recombinant Lactococcus lactis capable of secreting laccase and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of the recombinant Lactococcus lactis with exocrine laccase ability

[0048] 1.1 Design and synthesis of CotA laccase gene and construction of pUC57-Simple-S-CotA recombinant cloning vector

[0049] The Bacillus subtilis CotA laccase mature peptide was obtained through GenBank (accession number: FJ663050.1). The Usp45 secretion signal peptide and His tag were designed to be added to the CotA laccase sequence. According to the selection of the restriction site on the lactic acid bacteria expression vector pMG36e, Xma I restriction site and Hind III restriction restriction were added to the N-terminal and C-terminal of the protein sequence respectively. site. The designed sequence is optimized according to the codon preference of Lactococcus lactis, and the optimized sequence is shown in SEQ ID NO.3. The optimized CotA laccase sequence containing the Usp45 secretion signal peptide was named S-CotA. The optimized CotA laccase sequence was entruste...

Embodiment 2

[0090] Example 2 PCR amplification of CotA laccase gene

[0091] 2.1 Extraction of pUC57-Simple-S-CotA recombinant plasmid

[0092] The pUC57-Simple-S-CotA recombinant cloning vector was constructed and transformed into E.coli DH5α, the recombinant strain was activated on LB solid medium containing 100 μg / mL ampicillin (Ampr), and the plasmid mini-kit (EasyPureHiPure Plasmid) was used to activate the recombinant strain. MiniPrep Kit) to extract the pUC57-Simple-S-CotA recombinant plasmid. The specific operation steps are the same as the operation steps of 1.2.2 to extract the pMG36e-S-CotA recombinant plasmid with the EasyPure HiPure Plasmid MiniPrep Kit.

[0093] 2.2 Primer design of CotA laccase gene

[0094] According to the base sequence of the laccase gene in Bacillus subtilis after codon optimization (without Usp45 signal peptide) and the selection of the restriction enzyme cleavage site on the E. coli expression vector pET-30a(+), primers were designed by Primer 5.0 so...

Embodiment 3

[0106] Example 3 Identification of pMG36e-S-CotA recombinant expression vector

[0107] 3.1 PCR identification of positive clones

[0108] 1) Primer design

[0109] The primers were designed and identified according to the sequences on the pMG36e vector, and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The synthetic primers are shown in Table 4.

[0110] Table 4 Primer sequences

[0111]

[0112]

[0113] 2) PCR reaction system

[0114] The PCR reaction system and reaction conditions are shown in Table 5 and Table 6. After PCR, the PCR results were detected by 1% agarose gel electrophoresis.

[0115] Table 5 PCR reaction system

[0116]

[0117] Table 6 PCR reaction conditions

[0118]

[0119] 3) PCR identification results

[0120] The result is as image 3 As shown, the size of the amplified band is about 1900bp, which is consistent with the size of the expected band (1942bp), which preliminarily proves that the recombinant e...

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Abstract

The invention provides a recombinant Lactococcus lactis capable of secreting laccase and its construction method and application. The present invention obtains the Bacillus subtilis CotA laccase gene sequence through GenBank, and according to the codon preference of Lactococcus lactis NZ9000 will contain The CotA laccase gene of Usp45 signal peptide was codon-optimized, and then the codon-optimized CotA laccase gene was connected to the pMG36e vector, and electrotransformed into Lactococcus lactis NZ9000 to construct a recombinant lactic acid bacterium Lactococcus with the ability to secrete laccase . The extracellular laccase production of this strain is relatively high, which can increase silage efficiency and improve the quality of silage straw. It is a silage lactic acid bacterium with potential application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of enzymes, and in particular relates to a recombinant Lactococcus lactis with exocrine laccase ability and a construction method and application thereof. Background technique [0002] Silage is a method in which green feed is formed by anaerobic fermentation of lactic acid bacteria in a closed environment to form a palatability, rich nutrition, storage resistance and can maintain various nutritional properties of green feed all year round. Silage is an important material basis for the development of animal husbandry. With the rapid development of animal husbandry in my country, the problem of shortage of forage for herbivorous livestock is becoming more and more serious. Improving the utilization rate of feed is the key to solving the shortage of feed. Lactic acid bacteria determine the quality of silage. However, the number of lactic acid bacteria attached to natural forage grass is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/53A23K30/18C12R1/01
CPCC12N9/0061C12Y110/03002C12N15/746A23K30/18C12N2800/22C07K2319/02C07K2319/21A23V2400/231
Inventor 李冠华汪露露孔祥婕
Owner INNER MONGOLIA UNIVERSITY
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