A method for enzymatically preparing xylo-oligosaccharides
A technology of xylooligosaccharide and enzymatic preparation, which is applied in the field of genetic engineering and can solve the problems of undiscovered xylooligosaccharide
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Embodiment 1
[0025] Example 1: Obtaining the target gene.
[0026] Primers were designed according to the EG I gene (M15665.1) of Trichoderma reesei on NCBI to obtain the endoglucanase (EG I) gene (its nucleotide sequence is shown in SEQ ID NO: 1), the primer The design software adopts Stratagene's online primer design software Primer Design Program. EcoR I and Not I restriction sites were selected, and the primers were designed as follows (the restriction sites are underlined):
[0027] c.boipp.f (SEQ ID NO: 2) 5'— GAATTC ATGGCGCCCTCAGTTACAC—3'
[0028] EcoR I
[0029] c.boitt.r (SEQ ID NO: 3) 5'— GCGGCCGC TCAACGCTCTAAAGGCAT—3'
[0030] Not I
[0031] Table 1 PCR reaction parameters
[0032]
[0033] The target gene was obtained by PCR, and the gene is shown in SEQ ID No: 1, and the specific conditions are shown in Table 1.
Embodiment 2
[0034] Example 2: Construction of recombinant plasmids.
[0035] The recombinant plasmid pPICZαA-eg I was constructed (for details, refer to the Mulit-Copy PichiaExpression Kit operating manual of Invitrogen Company). Since the primers designed for PCR have EcoR I and Not I restriction sites, the target obtained by PCR The gene should also have two corresponding restriction sites at both ends (confirmed after sequencing), the target gene is amplified and cut out the sticky ends of the EcoR I and Not I restriction sites with restriction endonucleases At the same time, the corresponding cohesive ends of the pPICZαA plasmid were cut out with the same restriction enzymes, and then the two were ligated with T4 ligase, transduced into Escherichia coli, amplified and screened to obtain pPICZαA-eg I recombinant Plasmid, whose structure is as figure 1 As shown, the plasmid is a shuttle plasmid, which is then used for electroporation of host bacteria after linearization.
Embodiment 3
[0036] Example 3: Preparation of target DNA and electroporation transformation of Pichia pastoris.
[0037] The obtained recombinant plasmid was amplified in Escherichia coli, and the plasmid was extracted, and digested with Pme I endonuclease (specific conditions are shown in Table 2) to obtain linearized target DNA; preparation of competent cells: inoculate Pichia pastoris Transfer single colony X33 to a shaking tube containing 5mL YPD liquid medium, cultivate overnight at 30°C, 220r / min, then transfer to a 1L Erlenmeyer flask containing 250mL YPD liquid medium, inoculum 1%, cultivate overnight at 30°C, 20r / min , until OD600=1.36; centrifuge the culture medium at 1500g for 5min at 4°C, then resuspend the cells with 250mL ice-bathed sterile water; centrifuge at 1500g for 5min at 4°C, and then resuspend the cells with 125mL ice-bathed sterile water; Centrifuge at 1500g for 5min at 4°C, then resuspend the cells with 20mL of 1M sorbitol solution in ice bath; The volume is appro...
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