Difunctional enzyme with endoglucanase/xylanase and preparation method and application thereof

A technology for the activity of endoglucanase and xylanase, which is applied in the field of bioengineering and can solve the problems of difficult conversion and utilization, and difficult degradation of cellulose.

Active Publication Date: 2010-12-08
FUDAN UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

Lignocellulose consists of hemicellulose (about 20%-30%), cellulose (about 40%) and lignin (about 20%-30%) (Wongetal., Microbiol.Rev, 1988, 52 : 305-317), the microfibrils formed by the aggregatio

Method used

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  • Difunctional enzyme with endoglucanase/xylanase and preparation method and application thereof
  • Difunctional enzyme with endoglucanase/xylanase and preparation method and application thereof
  • Difunctional enzyme with endoglucanase/xylanase and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 The construction of Chinese yak rumen microbial metagenomic DNA library

[0030] The rumen of Chinese Qinghai yaks in Xining City was collected and filtered with 3 layers of gauze. The filtrate was centrifuged to collect rumen microbial cells and stored at -80°C until use. Take 100-200μl bacterial sample, wash 2-3 times with 1ml PBS, add 650μl DNA extraction buffer (Tris-HCl, 100mMpH8.0; Na 2 EDTA100mMpH8.0; Na 3 PO 4 Buffer100mMpH8.0; NaCl1.5M; CTAB1%; pH8.0), after mixing, place at -80°C, then place in a 65°C water bath to melt, repeat three times; add 3-4μL lysozyme (100mg / L) after cooling Shake horizontally on a shaker (37°C, 225rpm) for about 30min; add 2-3μL of proteinase K (20mg / mL) and continue shaking for about 30min; add 50-70μl of SDS (20%), mix well, and incubate at 65°C for 1- 2h, mix the centrifuge tube up and down every 10-20min; centrifuge at 12,000rpm room temperature for 10min, collect the supernatant, add 400-500μl of phenol:chloroform:is...

Embodiment 2c

[0032] Example 2 Screening of cosmid library endoglucanase

[0033] Amplification of the library and preparation of cell lysate: Inoculate a copy of the library formatted and stored in a 96-well plate into a 96-well plate containing 200 μl LB-Amp medium / well, culture at 37°C for 12 hours, and collect cells by centrifugation. Add 20 μl of phosphate-citrate buffer (pH4.8) / well to suspend the cells, place at -80°C for 15min, then place the cells in a 37°C water bath to melt, and repeat three times.

[0034] The screening of endoglucanase adopts sodium carboxymethylcellulose (CMC)-Congo red staining and decolorization method (Teatheretal., ApplEnvironMicrobiol, 1982, 43 (4): 777-780), take 10 μ l of the cell lysate prepared above Point on the agarose plate containing 0.5% CMC, place it at 37°C for 1 hour, and then use ddH 2 O Wash the plate to remove the residual bacterial solution, stain with 1% Congo red solution for 10 min, and then decolorize with 1M NaCl solution. The librar...

Embodiment 3

[0035] Cloning and sequence analysis of embodiment 3 RuCelA ​​gene

[0036] The above-mentioned endoglucanase-containing gene was cloned onto the pGEMllz vector by subcloning: the cosmid plasmid of the positive clone was partially digested with Sau3AI into a 2-5kb fragment, and connected into a BamHI digested and dephosphorylated In the pGEMllz vector, transform Escherichia coli DH5α, perform functional screening on the subcloning library by the method described in Example 2, sequence the obtained subclones with T7 and SP6 universal primers, and determine the endoglucanase by homologous comparison The coding region sequence of the gene, its gene nucleotide sequence is shown in SEQIDNO1, and named as RuCelA.

[0037] The gene cds encodes 551 amino acids, the ORF sequence is shown in SEQ ID NO2, and the theoretical molecular weight is about 60kD. Using SMART to analyze the structural domain, it was shown that the 21 amino acids from the N-terminal were the signal peptide sequen...

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Abstract

The invention belongs to the technical field of biological engineering, in particular to a difunctional enzyme RuCelA with endoglucanase/xylanase and a preparation method and application thereof. The RuCelA sources from uncultured microorganism of rumen of Chinese yaks, and has a certain activity on phosphocellulose and filter. The RuCelA not only can hydrolyze polysaccharide but also can hydrolyze trisaccharide to obtain monosaccharide; meanwhile, the RuCelA has synergistic effect with xylanase to synergetically degrade xylan; and the RuCelA can be utilized to realize mono enzymatic degradation on preprocessed lignocellulose. The difunctional enzyme RuCelA of the invention can be widely applied to cellulose degradation, comprising cellulose biotransformation, chemical industry, spinning, food, bioenergy, feed ingredients, pharmaceutical Industry and other aspects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a new bifunctional cellulose degrading enzyme RuCelA ​​with endoglucanase and xylanase activities, its preparation method and application. Background technique [0002] With the increasing shortage of energy in the world, the development of renewable bio-energy has attracted more and more attention. Lignocellulose is the most abundant renewable resource in nature. Cellulase and hemicellulase can be used to degrade plant fibers into fermentable sugars. However, lignocellulose is difficult to degrade under natural conditions, which is due to its chemical composition. related to structure. Lignocellulose consists of hemicellulose (about 20%-30%), cellulose (about 40%) and lignin (about 20%-30%) (Wongetal., Microbiol.Rev, 1988, 52 : 305-317), the microfibrils formed by the aggregation of cellulose molecules are embedded between hemicellulose and lignin to form a network struct...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12P19/14
Inventor 吕红常磊周峻岗
Owner FUDAN UNIV
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