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Construction of yeast strain with dual functions of producing and recycling cellulose excision enzyme

A yeast strain, dual-function technology, applied in the field of enzyme engineering, can solve the problems of increasing the production cost of cellulase, loss of activity, etc., and achieve the effects of reducing energy consumption, high innovation, and cost reduction

Active Publication Date: 2014-02-19
中农华威生物制药(湖北)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of cellulose evaporation concentration or spray drying, part of the activity of cellulase is lost, and the concentration process greatly increases the production cost of cellulase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] (1) Extraction of pPIC9K plasmid

[0025] 1) Add 1% E. coli cells containing pPIC9K plasmid to 2 mL of LB medium.

[0026] 2) Shake culture at 37 °C for 12 h.

[0027] 3) Take 1.5 mL of bacterial solution into an EP tube, centrifuge at 4000 rpm for 3 min, and discard the supernatant.

[0028] 4) Add 0.1 mL of solution I (1% glucose, 50 mM EDTA pH 8.0, 25 mM Tris-HCl pH 8.0) and mix well.

[0029] 5) Add 0.2 mL of solution II (0.2 mM NaOH, 1% SDS), gently invert and mix, and place in an ice bath for 5 min.

[0030] 6) Add 0.15 mL of pre-cooled solution III (5 mol / L KAc, pH 4.8), gently invert and mix, and ice bath for 5 min.

[0031] 7) Centrifuge at 10,000 rpm for 20 min, and transfer the supernatant to another new EP tube.

[0032] 8) Add an equal volume of isoamyl alcohol, mix well and let stand for 10 min.

[0033] 9) Centrifuge at 10,000 rpm for 20 min and discard the supernatant.

[0034] 10) Wash once with 0.5 mL of 70% ethanol and drain all liquid.

[0035...

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PUM

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Abstract

The invention discloses construction of a yeast strain with dual functions of producing and recycling cellulose excision enzyme, and belongs to the field of enzyme engineering. The construction method comprises synthesizing the promoter sequence of saccharomyces cerevisiae TDH3, the coding sequence of signal peptide secreted by saccharomyces cerevisiae, the cellulose excision enzyme coding sequence of aspergillus fumigatus, the coding sequences of 400 amino acid fragments on C terminal of clumping factor of saccharomyces cerevisiae, and the terminator sequence of Saccharomyces cerevisiae TDH3 in sequence; adding restriction endonuclease sites on two terminals; constructing to a pPIC9K plasmid by digestion; transforming the plasmid into the yeast to obtain the yeast strain with dual functions of producing and recycling cellulose excision enzyme. The yield of cellulose excision enzyme is up to 86%; the enzyme activity of cellulose excision enzyme reaches 1.5U / g after fermentation. The yeast strain provided by the invention can complete production and synchronous concentration as well as recovery of cellulose excision enzyme in one step, can reduce energy consumption and lower the cost, and has high practicability.

Description

[0001] technical field [0002] The invention belongs to the field of enzyme engineering, in particular to the construction of a yeast strain with dual functions of producing and recovering cellulose exonuclease. Background technique [0003] Cellulose is the most abundant renewable resource on earth. The amount of cellulose produced by photosynthesis on earth reaches about 10 billion tons each year. The use of cellulose to produce bioenergy is the key to alleviating the energy crisis and realizing sustainable human development. [0004] The key to cellulose utilization is to degrade cellulose into fermentable sugar-glucose. The degradation of cellulose requires the combined action of exonuclease, endonuclease and glucanosidase. Among them, exonuclease degrades the crystalline structure of cellulose, which is the rate-limiting step of cellulose degradation. Degradation of cellulose with cellulase has the characteristics of green, mild conditions and high conversion rate, b...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/81C12N1/19C12R1/865C12R1/68
Inventor 薛栋升汪江波蔡凤娇曹敬华陈茂彬方尚玲镇达
Owner 中农华威生物制药(湖北)有限公司
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