High-temperature alkaline pectate lyase Pel-863 and coding gene and application thereof
A technology of pectate lyase, pel-863, applied in the field of genetic engineering and biomass utilization, can solve the problems of fiber damage, environmental pollution and so on
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Embodiment 1
[0030] Embodiment 1: Construction of engineering bacteria containing high-temperature alkaline pectate lyase Pel-863 gene
[0031] 1.1 Extraction of Genomic DNA of C. kronotskyensis
[0032] Genomic DNA of C. kronotskyensis was extracted using the Bacterial Genomic DNA Extraction Kit (Tiangen Biotech), and stored at -20°C for future use.
[0033] 1.2 PCR amplification of Pel-863 gene and vector pET-28b
[0034] According to the published C. kronotskyensis genome information and its annotations, the primers for amplifying the pectate lyase gene were designed as follows:
[0035] Calkro_0863-F 5'-GCCGCGCGGCAGCATGGCGACACTTTTAACA-3'
[0036] Calkro_0863-R 5'-GCGGCCGCAAGCGTTTAGTATTGATGTATCTGTG-3'
[0037] Using the extracted genomic DNA as a template, PCR amplification of the target gene was carried out.
[0038] The vector pET-28b was digested overnight by Hind III and Nhe I, then subjected to agarose gel electrophoresis, and a fragment of about 5.3 kb in size was recovered us...
Embodiment 2
[0048] Example 2: Induced expression and purification of recombinant high-temperature alkaline pectate lyase Pel-863
[0049] The engineered bacteria obtained in Example 1 were cultured in LB liquid medium containing 50 μg / mL kanamycin, shaken at 200 rpm at 37°C until OD 600 When it reaches about 0.6, add IPTG to a final concentration of 0.1 mM, shake at 200 rpm at 37°C for 4-6 hours. The cells were collected by centrifugation at 4000 g for 15 min, resuspended in Binding Buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl), and sonicated in an ice bath. Then, centrifuge at 10,000g at 4°C for 15 minutes to remove cell debris, and the obtained supernatant is the crude enzyme solution.
[0050] The crude enzyme solution was heat-treated at 60°C for 30 minutes to remove heat-labile proteins, and centrifuged at 10,000 g at 4°C for 15 minutes to retain the supernatant. After equilibrating 600μL Ni-NAT affinity chromatography column with 6mL Binding Buffer, pass the supernatant through the...
Embodiment 3
[0051] Example 3: Determination of Enzymatic Properties of Recombinant High Temperature Alkaline Pectate Lyase Pel-863
[0052] 3.1 Determination of high temperature alkaline pectate lyase Pel-863 activity
[0053] The pectate lyase Pel-863 activity was analyzed by UV spectrophotometry: 80 μL pH7.0, 2% (W / V) PGA-NaOH solution, 320 μL Pel-863 buffer (50 mM glycine-hydrogen Sodium oxide, pH9.0, 150mM NaCl, 1.5mM CaCl 2 ), add 10 μL of appropriately diluted purified enzyme solution, react at 70° C. for 10 min, add 400 μL of 50 mM HCl to terminate the reaction, centrifuge the supernatant and measure the UV absorbance at 235 nm. A group without enzyme solution was used as a control. One enzyme activity unit (U) is defined as the amount of enzyme that produces 1 μmol of unsaturated galacturonic acid per minute under given conditions, where the molar absorptivity of unsaturated oligogalacturonic acid ε 235 4075M -1 cm -1 .
[0054] 3.2 Determination of optimal pH and pH stabili...
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