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Cracking polysaccharide monooxygenase and application thereof

A polysaccharide monooxygenase and enzymatic hydrolysis technology, applied in the field of bioengineering, can solve the problems of difficult and efficient hydrolysis, unfriendly environment, high energy consumption, etc.

Pending Publication Date: 2022-04-29
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, their recalcitrant crystalline structure makes them difficult to be efficiently hydrolyzed
Although chemical or physical methods can be used for pretreatment to reduce the crystallinity of the substrate, high energy consumption and unfriendly environment limit its large-scale industrial application.

Method used

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  • Cracking polysaccharide monooxygenase and application thereof
  • Cracking polysaccharide monooxygenase and application thereof
  • Cracking polysaccharide monooxygenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: the acquisition of lytic polysaccharide monooxygenase PlLPMOCB3 target gene

[0052] (1) The target gene was synthesized by Jinweizhi Company according to SEQIDNO.2, and 5'(EcoRI) and 3'(EcoRI) restriction sites were added during the synthesis, and the gene was cloned into the vector pET-28a through 5'EcoRI and 3'EcoRI (+) Construction of recombinant plasmids. The nucleotide sequences of the upstream and downstream primers are shown below:

[0053] Upstream primer:

[0054] 5'-ATGGGTCGCGGATCCATGATTCAAAACGTTCACGTTCAA-3';

[0055] Downstream primers:

[0056] 5'-TTGTCGACGGAGCTCGAATTCTTAATGGTGGTGGTGATGATG-3';

[0057] Plasmid pET-28a(+) was linearized, and the linearized plasmid was purified by DNA Extraction Mini Kit.

[0058] (2) Use T4 DNA ligase to connect the digested vector and the target base fragment together. The ligation system is as follows: 5 μL of the target gene, 3 μL of linearized vector, 1 μL of T4 DNA ligase, and 1 μL of Buffer. Ligatio...

Embodiment 2

[0060] Example 2: Construction and induction expression of recombinant Escherichia coli Rosetta-CB3

[0061] (1) Transformation of Escherichia coli. Take 10 μL of the recombinant pET-28a(+) plasmid constructed in Example 1 and add it to 80 μL of L. coliRosetta competent cells, mix gently and place on ice for 30 min, heat shock in a water bath at 42°C for 90 s, and immediately ice bath for 2-5 min. Add 1 mL of LB medium without resistance, resume culture at 220 rpm and 37 °C for 1 h, centrifuge to take 100-200 μL of bacterial liquid and spread it on LB solid medium containing kanamycin and chloramphenicol resistance, invert at 37 °C Incubate overnight. Pick a single colony on the LB plate into 5 mL of LB liquid medium containing kanamycin and chloramphenicol resistance, and cultivate at 37 ° C and 220 rpm for 12 h. Using the bacterial liquid as a template, PCR was performed at 1% (w / v ) of agarose gel electrophoresis verification results, screened positive clones for verifica...

Embodiment 3

[0065] Example 3: Substrate specificity analysis of PlLPMOCB3

[0066] Take several 1.5mL centrifuge tubes, add 1% (w / v) final concentration of colloidal chitin, chitosan, microcrystalline cellulose, CMC, corncob powder (pulverized and dried), filter paper, Beech wood xylan, add purified PlLPMOCB3 to make the final concentration 1 mg / mL, add ascorbic acid to make the final concentration 1 mM, use 50 mM, pH 5.0 sodium citrate buffer solution to accurately make up the volume to 1 mL, seal with parafilm Incubate at 50°C, shake at 220rpm for 36h, heat in boiling water for 5min to terminate the reaction, centrifuge at 12000rpm for 1min to collect the supernatant, and measure the yield of reducing sugar by PAHBAH method. The reaction system without enzyme was used as a blank control, and all samples were replicated 3 times each.

[0067] The result is as image 3 As shown, PlLPMOCB3 can act on colloidal chitin, chitosan, microcrystalline cellulose, CMC, corncob, filter paper and b...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a bacterial-derived lytic polysaccharide monooxygenase and application thereof. The amino acid sequence of the PlLPMOCB3 is as shown in SEQ ID NO. 1 in a sequence table. The PlLPMOCB3 not only has the capability of degrading biomass polysaccharides such as cellulose, xylan, chitin and the like, but also has a synergistic effect with commercial cellulase, xylanase or chitinase to improve the degradation efficiency of cellulose, lignocellulose, xylan and chitin substrates. Wherein when PlLPMOCB3 and commercial cellulase act together, the synergy degree of cellulose and corncob substrates can reach 1.43 and 1.22 respectively; when the xylanase acts together, the synergy degree reaches 4.57; and the synergism degree is up to 1.70 when the bacillus subtilis and chitinase act together. And a new way is provided for enriching a lysable polysaccharide monooxygenase library and artificially compounding a biomass degradation efficient compound enzyme system.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, in particular to a bacterial-derived lytic polysaccharide monooxygenase and its application. Background technique: [0002] Cellulose and chitin are the most abundant natural polysaccharides on earth and are the main production materials and renewable energy sources to replace fossil resources in the future. As natural macromolecular polymers, they need to be degraded into oligomers or monomers before they can be used effectively. At present, glycoside hydrolase system is mainly used to complete the catalytic degradation of biomass polysaccharides. However, their recalcitrant crystalline structure makes it difficult to be hydrolyzed efficiently. Although chemical or physical methods can be used for pretreatment to reduce substrate crystallinity, high energy consumption and unfriendly environment limit its large-scale industrial application. In order to improve the enzymatic hydrolysis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12P19/14C12P19/02C12P19/26
CPCC12N9/0083C12P19/14C12P19/02C12P19/26Y02E50/10
Inventor 马立娟凡林林张安琪王爽杜丽平张翠英肖冬光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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