Aspergillus niger recombinant strain capable of massively expressing Candida antarctica lipase B as well as construction method and application of aspergillus niger recombinant strain

A construction method and lipase technology are applied in the construction and application fields of high-yielding lipase B recombinant expression strains of Aspergillus niger, which can solve the problems of low expression and achieve the effect of high-efficiency secretory expression.

Pending Publication Date: 2021-12-03
南京正扬生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently reported research on the expression of lipase B using Aspergillus niger as a host is still very low (please refer to: 2019 literature "Secretary expression of lipase B from Candida antarctica in Aspergillus niger and its diatomaceous earth Fixed application")

Method used

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  • Aspergillus niger recombinant strain capable of massively expressing Candida antarctica lipase B as well as construction method and application of aspergillus niger recombinant strain
  • Aspergillus niger recombinant strain capable of massively expressing Candida antarctica lipase B as well as construction method and application of aspergillus niger recombinant strain
  • Aspergillus niger recombinant strain capable of massively expressing Candida antarctica lipase B as well as construction method and application of aspergillus niger recombinant strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Construction of p-ZYAN02-CALB plasmid

[0032] The preparation of the plasmid mainly includes the following two steps: 1. Preparation of the intermediate plasmid p-ZYAN01. 2. Linearization of the intermediate plasmid p-ZYAN01, and integration of the lipase B gene expression cassette and upstream and downstream homologous fragments into p-ZYAN01 to form p-ZYAN02-CALB plasmid

[0033] The method for preparing the intermediate plasmid p-ZYAN01 is as follows:

[0034] p-ZYAN01 is mainly composed of the following parts and the necessary connecting sequence, or directly combined with the following parts.

[0035] (1) 2305bp fragment obtained after XbaI-PscI double digestion of pUC57 plasmid;

[0036] (2) hyg gene expression cassette, the sequence is shown in SEQ ID NO.3;

[0037] (3) amds expression cassette, the sequence is shown in SEQ ID NO.4.

[0038] The 2305bp fragment obtained after pUC57 plasmid XbaI-PciI double digestion, the hyg gene expression cas...

Embodiment 2

[0039] Example 2: Transformation integration of lipase B expression cassette

[0040]The starting strain of this example is ZYAN05, which is obtained by knocking out the glucoamylase gene, fungal amylase gene and acid amylase gene from a conventional strain. The above-mentioned gene knockout / knock-in method in Aspergillus niger can be realized by referring to the technical methods disclosed in the examples of patent CN103937766A or CN104962594A. That is, refer to the method described by Delmas (Appl Environ Microbiol. 2014, 80(11): 3484-7) et al. Specifically, a circular DNA vector is used, which contains the above-mentioned 5' and 3' homologous sequences, a selectable marker, and an E. coli replication sequence. The circular vector was transformed into Aspergillus niger, and the recombinant strain was obtained by selection.

[0041] The p-ZYAN02-CALB plasmid is imported into Aspergillus niger strain ZYAN05 by the protoplast transformation method, and the specific operation ...

Embodiment 4

[0043] Embodiment 4 recombinant expression strain liquid fermentation produces lipase B

[0044] Slant culture: Take an inoculation loop of the bacterial lawn of the recombinant expression strain of Aspergillus niger and inoculate it on a PDA solid slope, and cultivate it at a constant temperature at 35°C for 60 hours; Shake flask culture: Take an inoculation loop of bacterial lawn from the strain obtained on the slope and insert it into the seeds for cultivation culture medium at an initial pH of 5.5, 35°C, and a shaker speed of 200 rpm for about 72 hours. The following table shows the fermentation enzyme production of 3 batches of 250ml shake flasks. The average enzyme production level is 10600unit / L of synthetic enzyme activity, and the crude protein content of the supernatant is 4g / L.

[0045] batch Fermentation period (h) Fermentation enzyme activity (unit / L) Crude protein (g / L) 1 72 9995 3.8g / L 2 78 12030 4.4g / L 3 75 9775 3.8g / L ...

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Abstract

The invention discloses an aspergillus niger recombinant expression strain of high-yield lipase B as well as a construction method and application of the aspergillus niger recombinant expression strain. According to the method for constructing the aspergillus niger recombinantexpression strain, a recombinant expression cassette containing the lipase B is constructed, and the recombinant expression cassette is a gene segment containing a lipase B gene sequence, a promoter, a terminator, a selection marker, upstream and downstream homologous sequences and other elements. The lipase B-producing aspergillus niger recombinant expression strain is constructed according to the method disclosed by the invention. The invention also discloses the application of the lipase B-producing aspergillus niger recombinant expression strain in expression of the lipase B. According to the invention, the expression cassette of the lipase B is constructed by means of genetic engineering, and the expression cassette is introduced into aspergillus niger expression host bacteria, so that efficient secretory expression of the lipase B is realized, and the high-yield lipase B expression strain is obtained. Through optimization of fermentation conditions, the expression level of supernatant crude enzyme liquid obtained through liquid fermentation of the strain can reach 4 g / L, and the synthetic enzyme activity of the lipase B can reach 10600unit / L.

Description

technical field [0001] The invention belongs to the field of genetic engineering breeding, and relates to the construction and application of a high-yield lipase B Aspergillus niger recombinant expression strain. Background technique [0002] Lipase B is a biocatalyst with dual lipid catalysis. First, lipase B can catalyze the hydrolysis of lipids into alcohols and fatty acids. Secondly, under certain conditions, lipase B can also catalyze the dehydration condensation of alcohols and fatty acids to generate lipids. At the same time, under certain conditions, it can also catalyze the transesterification reaction of lipids with corresponding acids or alcohols to generate new lipids. Therefore, it is different from conventional hydrolytic lipases. It is used in various industrial productions such as cheese manufacturing, lipid modification, lipid hydrolysis, pharmaceutical intermediate production, food processing, biodiesel and lubricating oil production. However, its relat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/55C12N15/80C12N9/20C12R1/685
CPCC12N9/20C12Y301/01003C12N15/80
Inventor 赵正阳
Owner 南京正扬生物科技有限公司
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