High-sugar-tolerant beta-glucosaccharase Bg14, and expressed gene and application thereof
A glucosidase and high sugar tolerance technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of intolerance of β-glucosidase, increased production cost of high-concentration glucose, etc.
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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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