Method for producing mutant β-glucosidase, enzyme composition for decomposing biomass, and sugar solution
A technology of beta glucosidase and enzyme composition, which is applied in the field of production of mutant beta glucosidase, enzyme composition for biomass decomposition and sugar liquid, can solve the problems of unclear relationship between functions and the like, and achieve glucosidase enzyme The effect of enzyme activity improvement
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[0081] First, as a method for preparing the above-mentioned DNA, it can be exemplified: a method of fully synthesizing a DNA encoding an amino acid sequence of interest by gene synthesis; 90%, 95%, 99%, or more of the amino acid sequence encoding the sequence number 1 A method of introducing a mutation at the above-mentioned predetermined position by site-directed mutagenesis in the DNA having an amino acid sequence having sequence identity. The gene encoding the amino acid sequence having 90%, 95%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 can be obtained from microorganisms retaining the β-glucosidase, especially Pyrococcus furiosus according to known Methods to separate DNA, using PCR and other techniques for DNA amplification, so as to obtain.
[0082] An expression vector comprising the DNA encoding the mutant β-glucosidase prepared as described above can be produced by ligating it downstream of a promoter in an appropriate expression vect...
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[0098] The following examples will be given to specifically describe the present invention. However, the present invention is not limited to these examples.
reference example 1
[0099] (Reference example 1) Preparation of pro-β-glucosidase by recombinant expression of Escherichia coli
[0100] The β-glucosidase-friendly gene was completely synthesized with the base sequence shown in SEQ ID NO: 2, ligated with NcoI and BamHI of pET-11d using Ligation High (Toyobo), and transformed into JM109 (Takara). Selection was performed using LB agar medium containing ampicillin as an antibiotic. The prepared vector pET-PfuBGL was isolated from the transformed JM109 strain using a mini-extraction kit (Kiagen), and base sequence analysis was performed. Escherichia coli BL21(DE3)pLysS strain for expression was transformed with pET-PfuBGL to prepare BL21-PfuBGL strain. The BL21-PfuBGL strain was inoculated in 10 mL of LB medium containing ampicillin, and cultured with shaking at 37° C. overnight (preculture). As the main culture, the bacterial cells obtained before inoculation were cultured in 1 L of ampicillin-containing LB medium, and cultured with shaking until ...
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