Secretory expression method for exoinulinase from Kluyveromyces marxianus

An exo-inulinase, secreted expression technology, applied in the field of exo-inulinase expression, 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: 2012-05-23
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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  • Application Information

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 sequence determination of Kluyveromyces marxii gene with signal peptide inulinase

[0029] Kluyveromyces marxianus K. marxianus CBS6556 genomic DNA was extracted with reference to the method described in "Refined Molecular Biology Experiment Guide" (Fourth Edition, Osper et al., Yan Ziying et al. Translated, published by Science Press), V- 530 ultraviolet / visible light / 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 the conventional method (molecular The third edition of Cloning Experiment Guide, written by Sam Brook, translated by Huang Peitang, 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-...

Embodiment 2

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

[0059] According to the two-terminal sequence of the mature protein encoded by the K.marxianus CBS6556 inulinase gene and the polyclonal restriction site sequence information of the Pichia pastoris expression vector pPICZαA (purchased from Invitrogen), a pair of specific primers were designed with the following sequences:

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

[0061] Inu-p2: 5'-CTC AAGGTTAAAATTGGGTAACGTT-3' (the single underlined part is the Xba I restriction sequence, and 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 coding of the inulinase mature peptide was amplified ...

Embodiment 3

[0120] Example 3: Transformation of Pichia pastorii strains and screening of positive clones

[0121] Recombinant expression plasmid inulinase was single-digested with sac I (Dalian TakaRa) and linearized, extracted with phenol: chloroform: isoamyl alcohol to remove protein, added 1 / 10 volume of 3mol / l sodium acetate solution (pH5.2) and 2 Double the volume of absolute ethanol, precipitate and recover the linearized carrier, and the concentration of the purified linearized carrier is 995 ng / μl as determined by a V-530 mol / l ultraviolet / visible light / near-infrared spectrometer (JASCO). 10 μl of the purified linear vector was taken and transformed into Pichia pastoris X-33 (purchased from Invitrogen) by electroporation, and transformed into the linearized pPICZαA empty vector as a control. Electric shock conversion parameters: Electric shock parameters: 0°C, 1.5kv, 200Ω, 25μF, electric shock time: 4.5-10ms, see the operation process instructions for the preparation of competent ...

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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 Kluyveromyces marx inulinase. Background technique [0002] Inulin is polyfructose, and there are about 30,000 kinds of plants in nature containing inulin polysaccharides. The annual output of inulin in the world can reach 350,000 tons, and it is the second largest plant carbohydrate after starch. Inulin-producing plants that are widely used in industry and as biomass raw materials are chicory and Jerusalem artichoke. These biomasses undergo physical (heating), chemical (acid hydrolysis) and enzymatic treatment to generate fructose and a small amount of glucose that can be utilized by various 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 Patents(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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