A mutant of alginate lyase based on loop region transformation and its application
A technology of alginate lyase and mutants, which is applied in the field of alginate lyase mutants, can solve the problems of uncertain effects, a large amount of experimental workload, etc., and achieve the effect of improving stability
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Embodiment 1
[0025] Embodiment 1: Fermentation culture condition and enzymatic assay method
[0026] Put the recombinant strain in LB liquid medium: peptone 10.0g / L, yeast powder 5.0g / L, NaCl 5.0g / L.
[0027] Determination of Alginate Lyase Enzyme Activity:
[0028] A slight adjustment is made on the improved DNS method. Add 0.1mL enzyme solution to 0.9mL 1% sodium alginate solution, mix well and react at 45°C for 20min, then add 1mL DNS to the system, stop the reaction in a boiling water bath for 3min, quickly dilute to 10mL, pipette 200μL into 96 wells plate, the absorbance was measured at 520 nm. In the control group, deionized water was used instead of crude enzyme solution. 1 Definition of Enzyme Activity Unit (U): Under the above conditions, the amount of enzyme required to produce 1 μg of reducing sugar per minute from 1 mL of enzyme solution. Prepare different concentrations of glucuronic acid solutions, add 1mL DNS reagent to react, and measure the absorbance of different conc...
Embodiment 2
[0029] Example 2: Simulation of crystal structure of alginate lyase derived from Escherichia coli
[0030] Using the reported alginate lyase AlyA5 alginate lyase (PDB code: 4BE3) as a template, the structure of alginate lyase whose sequence is SEQ ID NO.1 was constructed. The 201st aspartic acid to 211th tyrosine is the loop region near the catalytic domain of the alginate lyase in this study.
Embodiment 3
[0031] Example 3: Site-directed mutation at position 205 of the parental alginate lyase
[0032] Use the site-directed mutagenesis kit (TaKaRa), design primers 205Y-F, 205Y-R (as shown in Table 1), and use the constructed pET-28a(+) as a template to carry out PCR, and the 205th glycine of alginate lyase Replaced with tyrosine, the PCR reaction conditions were: 98°C for 5min, 34 cycles (98°C for 30S, 57.6°C for 60S, 72°C for 1.5min), 72°C for 10min. PCR amplification system: template 1 μL, upstream and downstream primers 1 μL each, Prime Star (Premix) DNA 20 μL, ddH2O 17 μL. The gel recovery kit was used to purify and recover the PCR product, and the concentration of the recovered product was checked by electrophoresis. Digest under the action of Bam HI and Mlu I fast-cutting enzymes, try to use T4 ligase method to ligate the PCR recovered product, transform it into competent E.coilJM109, spread ampicillin LB plate, and pick positive colonies. After culturing overnight on a s...
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