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High-sugar-tolerant beta-glucosaccharase Bg14, and expressed gene and application thereof

A technology for glucosidase and gene expression, which is applied in the field of cloning, recombinant expression and application of a novel high-glucose-tolerant β-glucosidase Bgl4 gene, and can solve the problem of β-glucosidase intolerance, increased production cost of high-concentration glucose, etc. question

Active Publication Date: 2014-12-10
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Technical problem to be solved: the technical problem mainly solved by the present invention lies in the problem that β-glucosidase cannot tolerate high concentration of glucose in the current cellulose degradation technology, which causes the problem of increased production cost, and then provides a high-sugar-resistant β-glucose Glucosidase Bgl4 and its expression gene and application

Method used

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  • High-sugar-tolerant beta-glucosaccharase Bg14, and expressed gene and application thereof
  • High-sugar-tolerant beta-glucosaccharase Bg14, and expressed gene and application thereof
  • High-sugar-tolerant beta-glucosaccharase Bg14, and expressed gene and application thereof

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Experimental program
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Embodiment 1

[0031] Example 1. Obtaining of total RNA from Aspergillus niger

[0032] 1.1 Cultivation of Aspergillus niger

[0033] Aspergillus niger can be screened from nature, or can be purchased from commercial channels (for example, it can be purchased from China Industrial Microorganism Culture Collection and Management Center).

[0034] The medium formula of Aspergillus niger strain (Aspergillus.niger) is: glucose 30g / L, K 2 HPO 4 ·3H 2 O 1g / L, KCl 0.5g / L, MgSO 4 0.5g / L, FeSO 4 0.01g / L, NaNO 3 0.2g / L, adjust the pH to 4.8, inoculate fresh Aspergillus niger spore suspension, culture at 180rpm at 37°C for 3-4 days and collect mycelium by filtration.

[0035] 1.2 Extraction of total RNA from Aspergillus niger

[0036]Take the collected mycelium of Aspergillus niger strain (Aspergillus.niger) and wash it once with PBS buffer solution, grind the mycelium under liquid nitrogen to powder, collect it into a 2mL centrifuge tube, add 1mL Trizol and shake vigorously. Centrifuge at 1200...

Embodiment 2

[0037] Cloning of embodiment 2.β-glucosidase Bgl4 coding gene

[0038] 1.1 Acquisition of Aspergillus.niger cDNA

[0039] Using the total RNA of Aspergillus niger strain as a template, the first strand of cDNA was synthesized by reverse transcription (the following reagents for reverse transcription were all from the kit "PrimeScriptTM1stStrand cDNA Synthesis Kit", purchased from Takara Company).

[0040] Prepare the following template RNA / Primer reactions in microcentrifuge tubes:

[0041] 50 μM Oligo dT 1 μL,

[0042] 10mM dNTP Mixture 1μL,

[0043] Total RNA 1 μg,

[0044] DEPC-H 2 O 7 μL;

[0045] After mixing, incubate at 65°C for 5 minutes and place on ice for 1 minute

[0046] Prepare the following cDNA synthesis reactions in the above microcentrifuge tubes:

[0047] 10 μL of the above RNA / Primer mixture,

[0048] 5×PrimeScript Buffer 4μL,

[0049] 40U / μL RNase Inhibiter 0.5μL,

[0050] 200U / μL PrimeScript RTase 1μL

[0051] RNase free H 2 O 4.5 μL;

[0052...

Embodiment 3

[0056] Construction and expression of embodiment 3.β-glucosidase Bgl4 expression vector

[0057] 3.1 Construction of β-glucosidase Bgl4 expression vector

[0058] The expression vector plasmid used is pPICZαA, with α-Factor signal peptide, so the signal peptide of the original gene needs to be removed, so the signal peptide is predicted for the protein sequence translated by the obtained gene Bgl4, and the prediction program adopts the online program SignalP4.0 (http : / / www.cbs.dtu.dk / services / SignalP / ) to obtain the signal peptide of high glucose-resistant β-glucosidase Bgl4, and design the signal peptide removal primer P3: 5'-CC according to the prediction results GGAATTC GCGCCTTCGTCGACCATCAAG-3', the underline indicates the EcoR I site, and primer P2 uses pMD-19T-Bgl4 as a template to amplify the Bgl4 fragment that removes the signal peptide. The PCR reaction conditions are 94°C, 5min; pause the timing, add Ex Taq polymerase, Add 40 μL of paraffin oil to seal; 35 cycles (...

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Abstract

The invention relates to high-sugar-tolerant beta-glucosaccharase Bg14, and an expressed gene and application thereof. The amino acid sequence is shown as SEQ ID NO.1. The gene of the high-sugar-tolerant beta-glucosaccharase Bg14 of aspergillusniger is cloned, recombined and expressed for the first time; and the glucose-tolerant coefficient Ki of the recombinase Bg14 is 2 mol / L.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to the cloning, recombinant expression and application of a novel high-glucose-resistant β-glucosidase Bgl4 gene derived from Aspergillus niger. Background technique [0002] β-glucosidase (β-glucosidase, EC 3.2.1.21) belongs to the class of exohydrolase, also known as β-D-glucoside hydrolase, is a class of enzymes that catalyze the transfer of glycosides between nucleophilic molecules, and can be used in short β-1,4-glucosidic bonds are broken between the residues of carbohydrates with aromatic groups or alkyl groups inside the chain of oligosaccharides or cellobiose, so they are widely used in the fields of cellulose hydrolysis (Science 311 :484-489.). As one of the main biological organisms produced by photosynthesis of green plants, cellulose, together with hemicellulose and lignin, serves as the supporting tissue of plants and is c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N15/66C12N1/19C12P19/14C12R1/84
Inventor 赵林果解静聪裴建军王飞庞倩
Owner NANJING FORESTRY UNIV
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