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Method of increasing yield of lignocellulose substrate hydrolase

A lignocellulose and cellulase technology, applied in the field of bioengineering, can solve the problems of reduced specific enzyme activity, large enzyme dosage, low enzymatic hydrolysis efficiency of cellulase system, etc., and achieves wide application value, increased copy number, high efficiency expressive effect

Inactive Publication Date: 2017-02-22
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these self-produced enzymes are born with certain ratio defects. As far as Trichoderma reesei (Trichoderma reesei), the most widely used cellulase-producing fungus, its cellulase system lacks exonuclease CBH II and β-glucosidase BG, resulting in low enzymatic hydrolysis efficiency of its cellulase system
In addition, whether it is a self-produced enzyme system derived from organisms or a commercial cellulase preparation made artificially, there are more than 80 protein types in it, resulting in a ratio of hemicellulase and cellulase core enzyme components. Reduced enzyme activity, resulting in inefficient hydrolysis of lignocellulose
More importantly, the proportions of cellulose, hemicellulose, and lignin in different lignocellulosic raw materials are significantly different, and the pretreated lignocellulose also has hemicellulose residues. Low hydrolysis efficiency and high enzyme dosage

Method used

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  • Method of increasing yield of lignocellulose substrate hydrolase
  • Method of increasing yield of lignocellulose substrate hydrolase
  • Method of increasing yield of lignocellulose substrate hydrolase

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Codon optimization of xylanase (XynC) gene

[0034] Search the Aspergillus niger xylanase gene sequence (accession number: KJ601783) in GenBank of NCBI (http: / / www.ncbi.nlm.nih.gov / ), and perform codon optimization according to the following method:

[0035] (1) Using the software GeneDesigner 2.0, the XynC codon (SEQ ID NO.1) was initially replaced according to Pichia pastoris codon preference; and the GC content was calculated, and then further adjusted according to the GC content to make the value within 40 ~50%. Analyze the distribution of hidden splice sites and codons of mRNA in the adjusted gene sequence. With the help of this software, the amount of change before and after codons can be visually analyzed.

[0036] (2) Using RNAStructure 3.2 to predict and analyze the free energy of the start codon end of the mRNA secondary structure, select a sequence with lower free energy and an open-loop structure at the start codon end, and finally obtain the optimized ...

Embodiment 2

[0039] Example 2: Construction of recombinant expression vectors pPIC9K-AxynC and pPICZαA-AxynC

[0040] The expression vectors pPIC9K and pPICZαA were digested with EcoR I and Not I, and the digested products were recovered. After purification, the synthesized target gene and the vector pPIC9K and pPICZαA use T 4 DNA ligase was used for ligation and transformed into Escherichia coli DH5α, and the target vectors pPIC9K-AxynC and pPICZαA-AxynC were obtained by colony PCR identification. Cultivate the Escherichia coli containing the target vector in LB medium for 12 to 16 hours, and extract the target vector according to the plasmid extraction manual. The construction and verification of expression vectors pPIC9K-AxynC and pPICZαA-AxynC are as follows figure 2 with image 3 Shown.

Embodiment 3

[0041] Example 3: Construction of a recombinant Pichia pastoris with high xylanase production and strain screening

[0042] Sac I was used to linearize pPIC9K-AxynC, and electroporation was used to transform Pichia pastoris GS115 competent cells. The transformation liquid was coated with RDB solid medium and cultured at 30°C for 3 days. Pick the colonies that grow normally and transfer them to YPD plates containing different concentrations (1, 2, 4, and 6 mg / mL) of geneticin G418, and pick the colonies that can grow normally on the high-concentration plates. The colonies obtained above were respectively inoculated into 250mL Erlenmeyer flasks of 30mL BMMY medium, cultured at 30°C and 220rpm for 4 days, and methanol was added every 24h to make the final concentration 0.5%. After the culture, the supernatant was collected by centrifugation. The enzyme activity was measured with beech xylan as the substrate, and the bacteria with the highest enzyme activity was No. 16 (named P.past...

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Abstract

The invention discloses provides a method of increasing heterologous expression quantity of hemicellulase, cellulase or lignocellulose hydrolysis assistance zymoprotein in pichia pastoris. An xylanase (XynC) gene from Aspergillus niger is subjected to codon optimization according to pichia pastoris codon preference characteristics, and an optimized nucleotide sequence (AxynC) is artificially synthesized, and constructs and recombines expression vectors pPIC9K-AxynC and pPICZ alpha A-AxynC. Recombined pichia pastoris of high yield xylanase is constructed by a two-step shock conversion method. That is to say, linearized pPIC9K-AxynC converts the pichia pastoris GS115 to obtain a strain with better enzyme producing ability. The high enzyme activity is subjected to second shock conversion as a host bacterium, and the linearized pPICZ alpha A-AxynC is shocked to convert the strain to obtain double-plasmid recombined pichia pastoris of the high yield xylanase. Tests show that the codon optimized xylanase gene can be expressed successfully in the pichia pastoris, the high yield xylanase recombinant bacterium constructed by a two-step shock conversion method can stably and efficiently produce the xylanase, the flask level enzyme activity reaches 410U / mL, and a protein content in a fermentated supernate reaches 0.37mg / mL.

Description

Technical field [0001] The invention relates to the technical field of bioengineering. In detail, the present invention relates to a method for increasing the expression level of hemicellulase, cellulase or auxiliary enzyme protein related to hydrolysis of lignocellulosic substrate in recombinant Pichia pastoris and the construction of double plasmid recombinant Pichia pastoris. Background technique [0002] Hemicellulase, cellulase, and auxiliary enzymes related to the hydrolysis of lignocellulosic substrates are the general term for a class of multi-component enzyme proteins, which can work together to hydrolyze lignocellulose that is difficult to be bioavailable into glucose that is beneficial to bioavailability. Therefore, it is considered as one of the enzyme preparations with great potential. Both hemicellulase and cellulase are glycoside hydrolases. The hemicellulase enzyme system capable of degrading lignocellulose includes β-D-1,4 endo-lignin which randomly cuts the xyl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N9/42C12R1/84
CPCC12N15/815C12N9/2482C12N2800/22C12Y302/01008
Inventor 孙付保张云博张震宇白仁惠杨慧敏王春迪
Owner JIANGNAN UNIV
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