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Novel high-activity allinase and preparation method thereof

An alliinase, high-activity technology, applied in the field of bioengineering, can solve problems such as difficult to obtain satisfactory results, and achieve the effects of simplifying production links and reducing production costs

Active Publication Date: 2016-08-10
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is generally difficult to obtain satisfactory results for a gene mutated once, so a sequential error-prone PCR (Sequential Error-prone PCR) strategy has been developed.

Method used

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  • Novel high-activity allinase and preparation method thereof
  • Novel high-activity allinase and preparation method thereof
  • Novel high-activity allinase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Obtaining the Mature Peptide Gene of Wild Type Alliinase

[0049] 1. Take fresh garlic bulbs and grind them in liquid nitrogen, extract total RNA according to the instructions of Trizol reagent and perform RT-PCR.

[0050] 2. Using the first strand of the synthesized cDNA as a template, design a pair of primers in the upstream and downstream of the ORF frame according to the alliinase sequence registered in Genbank sequence number S73324.1, and introduce restriction enzyme sites BamH I and Hind III respectively . Design the amplification primers of alliinase mature peptide gene of the present invention as follows:

[0051] Upstream primer P1 (SEQ ID NO.1): 5'-CGC GGATCC ATGATCTGCCTAGTGATTTTGACATG-3'

[0052] Downstream primer P2 (SEQ ID NO.2): 5'-CCC AAGCTT TTAAATGAAAGGACGACGGGAG-3'

[0053] The reaction conditions for its amplification are:

[0054]

[0055]

[0056] The amplification conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95...

Embodiment 2

[0058] Acquisition of a novel high-activity alliinase gene.

[0059] 1. The recombinant plasmid was transformed into Escherichia coli DH5α, and the wild-type alliinase gene was successfully cloned into the pET28a vector after verification by BamH I and Hind III double enzyme digestion.

[0060] 2. Random mutation

[0061] (1) Perform random mutation based on error-prone PCR technology to construct a new type of high-activity alliinase, and design primers as follows:

[0062] Upstream primer P1 (SEQ ID NO.1): 5'-CGC GGATCC ATGATCTGCCTAGTGATTTTGACATG-3'

[0063] Downstream primer P2 (SEQ ID NO.2): 5'-CCC AAGCTT TTAAATGAAAGGACGACGGGAG-3'

[0064] In the error-prone PCR reaction system, P1 and P2 were used as upstream and downstream primers, and the wild-type alliinase mature peptide gene was used as a template to perform error-prone PCR.

[0065] The reaction conditions for its amplification are:

[0066]

[0067]

[0068] The amplification conditions were: pre-dena...

Embodiment 3

[0084] Construction of Novel High-Active Alliinase Recombinant Bacteria of Bacillus subtilis

[0085] 1. Construction of expression vector pBSA43

[0086]pBSA43 is based on the Escherichia coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, cloned into a strong Bacillus constitutive promoter P43, and the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the medium . it comes with amp r Gene that can use ampicillin resistance as a selectable marker in E. coli; also has Km r Gene, kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis.

[0087] 2. Construction of a novel high-activity alliinase expression vector pBSA43-alliinasem

[0088] The novel high-activity alliinase gene obtained by error-prone PCR construction was double-digested with BamHI and HindIII and then ligated with the same double-digested Bacillus subtilis expression vector pBSA43 to construct the recomb...

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Abstract

The invention relates to a novel high-activity allinase and a preparation method thereof. The preparation method is characterized in that a wild type allinase gene from a garlic bulb is transformed by utilizing a random mutation technology, and a high-activity allinase mutant gene, namely a novel high-activity allinase gene, is obtained; then the novel high-activity allinase gene is expressed in a bacillus subtilis expression system and pichia pastoris expression systems (including a pichia pastoris free expression system and a pichia pastoris cell surface display system) respectively, and recombination strains for producing the high-activity allinase are obtained; detections carried out after fermentation expression show that the specific enzyme activity of the novel high-activity allinase is improved by 50% in comparison with a wild type allinase.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a novel alliinase mutant with improved specific enzyme activity constructed by in vitro directed evolution of error-prone PCR technology, in particular to random mutation of genes and recombinant DNA technology, especially a novel High-activity alliinase and its preparation method. technical background [0002] Alliinase, also known as alkyl cysteine ​​sulfoxide, C-S enzyme, etc. (E.C.4.4.1.4), was first discovered by Stoll and Seeback in 1949. It has dimer, trimer, tetramer and other forms. The aggregation forms of different plant proteins are different. For example, the alliinase of garlic (Alliium sativum) is a dimer, while that of onion (Alliium cepa) is a tetramer. [0003] Foreign scholars have obtained the gene encoding alliinase from onion, leek, bear onion and garlic, but there are many differences in the reported gene sequence, protein molecular weight and biochemical prop...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N15/10C12N9/88C12N15/75C12N15/81
CPCC12N9/88C12Y404/01004
Inventor 路福平刘逸寒郭伟韩杨贾雷博
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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