A genetic modification method for introducing polyamine tags, soluble expression and biomimetic immobilization of lipase

A genetic modification, lipase technology, applied in the field of protein immobilization and genetic engineering, can solve the problems of reducing enzyme utilization, inactivation, insolubility, etc., and achieve simple immobilization methods, high immobilization efficiency, and high vitality recovery. Effect

Active Publication Date: 2021-06-01
JILIN UNIV
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Problems solved by technology

Although later Jung et al. improved the expression of CalB in the periplasmic space through codon optimization and mutation of surface hydrophobic residues ("Improving the expression yield of Candida antarctica lipase B in Escherichiacoli by mutagenesis"), but still not completely Solving the problem of recombinant proteins existing in an insoluble form
[0004] Lipase is widely used, but there are usually some problems when using free natural enzymes directly to catalyze reactions, such as: free enzymes are insoluble in non-aqueous solvents and easy to aggregate, which greatly reduces the utilization rate of enzymes; Enzyme activity is unstable and easily inactivated by changes in external environmental factors (temperature, PH value, ionic strength); the separation of enzymes is difficult, and the enzymes involved in the reaction cannot be recycled and reused

Method used

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  • A genetic modification method for introducing polyamine tags, soluble expression and biomimetic immobilization of lipase
  • A genetic modification method for introducing polyamine tags, soluble expression and biomimetic immobilization of lipase
  • A genetic modification method for introducing polyamine tags, soluble expression and biomimetic immobilization of lipase

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: PCR method constructs and expresses the lipase gene 6His-CalB-10Lys with polyamine tag

[0047] Using PA1K-pPIC9K as a template, according to the known CalB gene sequence, design the upstream primer H6-CalB (F) (the underline is the NdeI restriction site) and the downstream primer CalB-K10 (R) (the underline is the XhoI restriction site ) sequence as follows:

[0048] H6-CalB(F):5'-ATATAT CATATG CACCACCCACCACCACCACCTACCTTCCGGTTCGGACCCTGC–3’

[0049] CalB-K10(R):5'-AATCGC CTCGAG TCATTTTTTTCTTTTTCTTTTTTTTCTTTTTCTTGGGGGTGACGATGCCG–3’

[0050] The plasmid PA1K-pPIC9K, upstream primers, downstream primers, deionized water, and DNA polymerase reaction mixture system were prepared according to the following volumes, and the lipase gene fragment 6His-CalB-10Lys rich in polyamine tags was amplified by PCR .

[0051]

[0052]

[0053] The PCR amplification program is: 94° C. for 2 min, 94° C. for 45 s, 55° C. for 45 s, and 72° C. for 2 min; a total of 35...

Embodiment 2

[0057] Embodiment 2: PCR method constructs and expresses the lipase gene 6His-CalB-10Arg with polyamine tag

[0058] The lipase CalB gene is derived from the plasmid PA1K-pPIC9K (Xiao Peiliang's master's thesis, the study of enzyme-catalyzed asymmetric synthesis of chiral caffeic acid derivatives), using PA1K-pPIC9K as a template, according to the known CAL-B gene sequence, design upstream primers The sequences of H6-CalB(F) (the underline is the NdeI restriction site) and the downstream primer CalB-R10(R) (the underline is the XhoI restriction site) are as follows:

[0059] H6-CalB(F):5'-ATATAT CATATG CACCACCCACCACCACCACCTACCTTCCGGTTCGGACCCTGC–3’

[0060] CalB-R10(R):5'-AATCGC CTCGAG TCAACGACGACGACGACGACGACGACGACGACGACGGGGGGTGACGATGCCG–3’

[0061] After the plasmid PA1K-pPIC9K, upstream primers, downstream primers, deionized water, and DNA polymerase reaction mixture system were prepared according to the following volumes, the polyamine-rich lipase gene fragment 6His-CalB-1...

Embodiment 3

[0067] Example 3: Expression plasmid transferred into BL21(DE3) competent cells for expression

[0068] The lipase expression plasmids 6His-CalB-10Lys-pET21a and 6His-CalB-10Arg-pET21a containing different polyamine tags prepared in Example 1 and Example 2 were respectively transferred into BL21 (DE3) competent cells, LB solid Medium plate, culture overnight, pick a single clone and inoculate it in LB liquid medium, cultivate to the logarithmic phase at 37°C; add 1mM IPTG, 16°C, 108rpm, cultivate for 14h, and obtain the expression strain BL21DE3 (6His-CalB-10Lys -pET21a) and the expression strain BL21DE3 (6His-CalB-10Arg-pET21a), induce the expression of the protein, that is, obtain the lipase containing the polyamine tag.

[0069] The amino acid sequence of 6His-CalB-10Lys lipase is:

[0070] MHHHHHHLPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVP...

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Abstract

The present invention provides a gene modification method to realize its soluble and active expression: take the lipase gene with polyamine tags at the N-terminus and C-terminus as the target gene, and add coding A nucleotide sequence of m histidines, m arginines or m lysines, m=2-10, and at the same time add n histidines and n sperms to the C-terminus of the lipase parent gene sequence amino acid or n lysine nucleotide sequence, n=2-10, and primers are designed for the target gene; the N-terminal and C-terminal lipase genes with polyamine tags are obtained by PCR. After the gene is expressed, a soluble lipase with a polyamine tag is produced, which can induce the formation and immobilization of biomimetic nano-silicon. The invention can obtain the immobilized enzyme with high activity and immobilization efficiency under mild conditions and in a short time; the operation is simple, the immobilization conditions are easy to control, and the cost is low.

Description

technical field [0001] The present invention relates to the technical fields of genetic engineering and protein immobilization. Specifically, the present invention relates to introducing a polyamine tag into the lipase gene by a genetic modification method, which can effectively realize the soluble expression of lipase in Escherichia coli and produce A lipase with a polycationic amino acid label; the lipase induces silicic acid to aggregate into silicon particles, and fixes proteins on the silicon particles through charge interaction and ion-interaction force, thereby obtaining the immobilized enzyme and its application. Background technique [0002] Lipase can catalyze esterification, hydrolysis, transesterification and acylation reactions. In non-aqueous phase catalyzed synthesis, many lipases, such as Candida antarctica Lipase B (CalB), exhibit excellent activity and operational stability; in organic synthesis, resolution of chiral compounds , pharmaceuticals, pharmaceut...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/70C12N11/14
CPCC07K2319/21C12N9/20C12N11/14C12N15/70C12Y301/01003
Inventor 郑良玉周小雪张锁秦田雪梅
Owner JILIN UNIV
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