Secretory expression method for exoinulinase from Kluyveromyces marxianus

A technology of exo-inulinase and expression method, which is applied in the field of expression of exo-inulinase, can solve the problems of inability to form natural inulinase molecules and affect the biological activity of recombinant inulinase, and achieve the goal of a good enzymatic tool Effect

Inactive Publication Date: 2009-07-01
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0008] After analysis, it was found that in the past when exogenous inulinase was expressed in Saccharomyces cerevisiae or Pichia pastoris, the natural N-terminus of the expression product was mostly blocked (the extra amino acid residues other than inulinase itself remained after the signal peptidase cleavage) ), the amino acid residues expressed by the carboxy-terminal residual vector sequence (patent application number: CN 02124145.7; Zhang LH, Wang J, Ohta Y, et al. Expression of the inulinasegene from Aspergillus niger in Pichia pastoris. Process Biochemistry 2003, 38: 1209 -1212.), unable to form natural inulinase molecules, may affect the biological activity of recombinant inulinase

Method used

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  • Secretory expression method for exoinulinase from Kluyveromyces marxianus
  • Secretory expression method for exoinulinase from Kluyveromyces marxianus
  • Secretory expression method for exoinulinase from Kluyveromyces marxianus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Cloning and sequencing of the signal peptide inulinase gene of Kluyveromyces marxianus

[0029] The genomic DNA of K. marxianus CBS6556 was extracted according to the method described in "Experimental Guide to Molecular Biology" (Fourth Edition, Osber et al., translated by Yan Ziying et al., published by Science Press), V- 530 UV / Visible / Near Infrared Spectrometer (JASCO) to measure its OD 260 / OD 280 Value=1.76, frozen at -20°C for later use. Using the genomic DNA of K.marxianus CBS6556 as a template, using two specific primers inu-ORF-p1: 5'-ATGAAGTTAGCATACTCCCTCTTGC-3' and inu-ORF-p2: 5'-TCAAAGGTTAAATTGGGTAACGTT-3', according to conventional methods (molecular The third edition of the cloning experiment guide, written by Sam Brook, translated by Huang Peitang, etc., published by Science Press) for polymerase chain reaction (PCR). PCR system: 10×PCR buffer (Dalian TakaRa) 5.0μl, dNTPs (10mmol / l, TaKaRa) 1.0μl, inu-ORF-p1 primer (10mmol / l) 2.0μl, inu-ORF-p2 prim...

Embodiment 2

[0058] Example 2: Construction of recombinant Pichia pastoris expression vector pPICZαA-inu12

[0059] According to the sequence information of the two ends of the mature protein encoded by the K. marxianus CBS6556 inulinase gene and the sequence information of the polyclonal restriction site of the Pichia expression vector pPICZαA (purchased from Invitrogen), a pair of specific primers was designed. The sequence is as follows:

[0060] Inu-p1: 5’-TC CTC GATGGTGACAGCAAGGCCAT-3' (the underlined part is the XhoI digestion sequence, and the boxed part is the coding sequence of the Kex2 signal peptidase recognition site)

[0061] Inu-p2: 5’-CTC TCTAGA AAGGTTAAATTGGGTAACGTT-3' (the single underlined part is the Xba I restriction sequence, the double underlined part is the stop codon sequence)

[0062] Using the T vector carrying the K. marxianus CBS6556 inulinase gene obtained in Example 1 as a template, using primers Inu-p1 and Inu-p2, the encoding of the mature peptide of inulina...

Embodiment 3

[0120] Example 3: Transformation of Pichia pastoris strain and screening of positive clones

[0121] Recombinant expression plasmid inulinase was linearized with sac I (Dalian TakaRa) single enzyme digestion, after phenol: chloroform: isoamyl alcohol extraction to remove protein, add 1 / 10 volume of 3mol / l sodium acetate solution (pH 5.2) and 2 Double the volume of absolute ethanol, precipitate and recover the linearized vector. The purified linear vector is measured by V-530mol / l ultraviolet / visible light / near infrared spectrometer (JASCO) to have a concentration of 995ng / μl. Take 10 μl of the purified linear vector and transform the Pichia pastoris X-33 (purchased from Invitrogen) by the electric shock method, and transform the linearized pPICZαA empty vector as a control at the same time. Electric shock transformation parameters: Electric shock parameters: 0℃, 1.5kv, 200Ω, 25μF, electric shock time: 4.5-10ms. For the preparation of competent cells, please refer to the operation ...

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Abstract

The invention relates to a eukaryon expression method for exoinulinase, which mainly comprises the following steps: firstly, connecting a nucleotide sequence (as shown in SEQ ID No: 1) of the exoinulinase of Kluyveromyces marxianus with pichia expression plasmids pPICZ alpha A through a restricted enzyme site, and obtaining a recombinant expression vector; and secondly, introducing the recombinant expression vector into a thermococcus host strain X-33 or SMD1168, and expressing target proteins (the Kluyveromyces marxianus exoinulinase) through induction fermentation. The eukaryon expression method for the exoinulinase lays a foundation for developing the exoinulinase with industrial application value.

Description

Technical field [0001] The invention relates to an expression method of exoinulinase (exo-D-fructosidase), in particular to a high-efficiency secretion expression method of inulinase of Kluyveromyces marxianus. Background technique [0002] Inulin is polyfructose. There are about 30,000 kinds of plants in nature that contain inulin polysaccharides. The world's annual output of inulin can reach 350,000 tons, which is the second largest plant carbohydrate after starch. Chicory and Jerusalem artichoke are the most commonly used inulin-producing plants in industry and as biomass raw materials. These biomasses undergo physical (heating), chemical (acid hydrolysis) and enzymatic treatments to produce fructose and a small amount of glucose that can be utilized by a variety of microorganisms (Bacon JSD, Edelmen J. The carbohydrates of the Jerusalem artichoke and other compositae. J. Biochem. 1951, 48: 114-126; Peters D. Carbohydrates for fermentation. Biotechnol J. 2006, 1(7-8): 806-814....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12R1/84
Inventor 张素芳赵宗保杨帆
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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