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Cellulase suitable for preparing cellulose nanocrystals, and application thereof

A cellulase, nanocrystal technology, applied in the biological field

Active Publication Date: 2020-04-17
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report about the cellulase suitable for preparing cellulose nanocrystals and the method of obtaining the cellulase by fermentation and rapidly preparing cellulose nanocrystals by constructing an engineering strain with a reasonable enzyme system

Method used

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  • Cellulase suitable for preparing cellulose nanocrystals, and application thereof
  • Cellulase suitable for preparing cellulose nanocrystals, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The construction of embodiment 1 engineering strain cEES

[0064] (1) Gene acquisition:

[0065]The activation of the cellulase gene (cbh1) (GenBank: GQ844299.1) was amplified with primers cbh1-U-F and cbh1-U-R using Penicillium oxalicum CGMCC No.5302 genome (GenBank: AGIH00000000.1) as a template Subsequence cbh1 (U), amplified with primers cbh1-D-hphR-F and cbh1-D-R to obtain the downstream sequence cbh1 (D) of the cellulase gene (cbh1) coding region, with primers Cel7B-cbh1 (U) R-F and Cel7B-hphF-R amplifies the gene fragment cel7B (GenBank: EU339127.1), and uses primers cel5B-F and cel5B-R to amplify the gene fragment cel5B (GenBank: KJ 955479); with Trichoderma reesei The cDNA of QM6a ATCC13631 (GenBank: AAIL00000000.2) was used as a template to amplify the gene swo1 (GenBank: AJ245918.1) with primers swo1-AgpdA(p)R-F and swo1-R; the plasmid pAN7-1 (GenBank: Z32698.1 ) was used as a template to amplify the promoter AgpdA( p)(GenBank:Z32524.1); the glyceraldehyde...

Embodiment 2

[0102] After the engineering strain cEES is activated, the seed liquid is obtained. Under sterile conditions, the hyphae are added to the fermentation medium in a proportion of 0.3% by weight, and cultured at 28°C and 250rpm for 7 days or in a fermenter for 7 days; Obtain the supernatant by centrifugation (10000rpm, 10min), which is the cellulase-containing fermentation broth. In the cellulase liquid, the ratio of endo-cellulase activity to exo-enzyme activity is 40, and the protein ratio of endo-cellulase Cel7B to Cel5B is 1.

[0103] Among them: the seed medium formula is: 20g / L glucose, Vogel's salt; the fermentation medium formula is: 10g / L bran, 10g / L microcrystalline cellulose, 3g / L KH 2 PO 4, 2.6g / L NaNO 3 , 0.5g / LMgSO 4 ·7H 2 O, 0.5g / L CaCl 2 , 1g / Lpeptone, 7.5mg / L FeSO 4 ·7H 2 O, 2.5mg / L MnSO 4 ·H 2 O, 3.6mg / L ZnSO 4 ·7H 2 O, 3.7mg / LCoCl 2 ·6H 2 O.

[0104] The formula of the above-mentioned Vogel's salt has been recorded and publicized in Chinese patent...

Embodiment 3

[0106] After the engineering strain cEES is activated, the seed liquid is obtained. Under sterile conditions, the hyphae are added to the fermentation medium in a proportion of 2% by weight, and cultivated at 35°C and 150rpm for 3 days or in a fermenter for 3 days; Obtain the supernatant by centrifugation (10000rpm, 10min), which is the cellulase-containing fermentation broth. In the cellulase liquid, the ratio of endo-cellulase activity to exo-enzyme activity was 44, and the protein ratio of endo-cellulase Cel7B to Cel5B was 1.1.

[0107] Among them: the seed medium formula is: 20g / L glucose, Vogel's salt; the fermentation medium formula is: 10g / L bran, 10g / L microcrystalline cellulose, 3g / L KH 2 PO 4 , 2.6g / L NaNO 3 , 0.5g / LMgSO 4 ·7H 2 O, 0.5g / L CaCl 2 , 1g / Lpeptone, 7.5mg / L FeSO 4 ·7H 2 O, 2.5mg / L MnSO 4 ·H 2 O, 3.6mg / L ZnSO 4 ·7H 2 O, 3.7mg / LCoCl 2 ·6H 2 O.

[0108] The formula of the above-mentioned Vogel's salt has been recorded and publicized in Chinese p...

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Abstract

The invention discloses a cellulase suitable for preparing cellulose nanocrystals, and an application thereof. The cellulase is prepared by the following steps: (1) constructing an engineering strain;and (2) producing the cellulase by liquid fermentation. An enzyme component for degrading a cellulose amorphous region and a protein component for depolymerizing hydrogen bonds in a large-size cellulose crystallization region are strengthened in the process of degrading cellulose with cellulase, so breakage of a cellulose macromolecular chain and a peeling reaction of the cellulose crystallization region are promoted, thereby cellulose crystals with the nanoscale can be easily formed by applying the cellulase. Compared with existing commercial cellulase, the cellulase disclosed by the invention has the following advantages: the enzyme system composition is more suitable for preparing the cellulose nanocrystals, the enzyme system is controllable, the hydrolysis efficiency is high, the enzyme cost is low, the preparation process is simple, the raw materials do not need to be subjected to mechanical treatment, the yield of the nanocellulose is high and the like.

Description

technical field [0001] The invention relates to a cellulase and an application thereof, in particular to a cellulase suitable for preparing cellulose nanocrystals and an application thereof, and belongs to the field of biotechnology. Background technique [0002] Cellulose nanocrystals have attracted extensive attention in recent years due to their renewable, nontoxic, high specific surface area, excellent mechanical properties, and easy functionalization of hydroxyl groups. Cellulose nanocrystals have the potential to develop a variety of products that are expected to replace traditional petrochemical and non-biodegradable polymer materials, including food packaging, films, and nanocellulose silver-loaded agents with antibacterial properties. [0003] The preparation methods of cellulose nanocrystals include concentrated acid hydrolysis and biological methods. Among them, the hydrolysis of concentrated sulfuric acid is the most commonly used preparation method at present, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12P19/14C12P19/04C12R1/80
CPCC12N9/2437C12N15/81C12P19/14C12P19/04
Inventor 赵建杨甜甜李雪芝
Owner SHANDONG UNIV
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