Pichia pastoris phaff recombined bacterium and uses thereof
A technology of recombinant bacteria and yeast, applied in the field of microorganisms, can solve problems such as environmental pollution in farms, and achieve the effects of improving brewing efficiency, high activity and reducing costs
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Embodiment 1
[0038] Embodiment 1, the preparation of Pichia pastoris recombinant strain X-33 / pPIC-glu-opt
[0039] 1. Optimum design of β-1,3-1,4-glucanase gene sequence
[0040]According to the β-1,3-1,4-glucanase gene sequence published in GenBank Accession Number EU082110, according to the codon preference of Pichia pastoris, the codons with high frequency of use were used to replace the codons with low frequency of use, Redesign the β-1,3-1,4-glucanase gene sequence and keep the amino acid sequence of the mature protein unchanged. To facilitate cloning, EcoRI and XbaI restriction sites were designed at the 5' and 3' ends of the sequence, respectively. The designed sequence was fully synthesized by Shanghai Sangon Bioengineering Co., Ltd. The optimized sequence is shown in sequence 1 in the sequence listing, and the amino acid sequence encoded by the nucleotide sequence is shown in sequence 2 in the sequence listing.
[0041] 2. Construction and transformation of Pichia pastoris expr...
Embodiment 2
[0046] Example 2. Production of β-1,3-1,4-glucanase by Pichia pastoris recombinant strain X-33 / pPIC-glu-opt
[0047] Under sterile conditions, inoculate the above-mentioned Pichia pastoris recombinant strain X-33 / pPIC-glu-opt in a 3 L shake flask containing 200 mL of BMGY medium, and shake at 28-30°C and 280rpm 12-24 hours, get OD 600 3.0 seed culture medium. Then prepare 4L of BMGY medium, put it into a 10L automatic control fermenter, sterilize it at 121°C, cool it to 28.5°C, add 200mL of the above seed culture solution, adjust the pH to 5.5 with ammonia water and phosphoric acid, adjust the rotation speed and air Flow control dissolved oxygen is greater than 20%. It was determined that 23 hours after the seed solution was inserted, the glycerol was completely consumed (dissolved oxygen was marked as 100%), and it entered the stage of feeding 25% (25g / 100ml) glycerol at a flow rate of 60mL / h. After 17 hours of feeding, the feeding of glycerin was stopped. After 3 hours, t...
Embodiment 3
[0061] Example 3, β-1,3-1,4-Glucanase enzymatic property analysis
[0062] 1. Optimum pH value and pH stability research
[0063] Determination of the optimum pH value: the dextran substrate was prepared with disodium hydrogen phosphate-citric acid buffer solution with pH values of 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, and 10.0, and the dextran substrate was prepared at 40 The enzyme activity was measured at the reaction temperature of ℃, and the optimum pH value of the enzyme was determined. The enzyme activity assay method is the same as in Example 2. The experiment was repeated three times, and the results are shown in Figure 4. The results showed that the catalytic pH range of the enzyme was relatively wide, from 5.4 to 7.4, and 6.4 was the optimum pH of the enzyme. The relative enzyme activity in Figure 4 is based on the enzyme activity at pH 6.4 as 100%.
[0064] Determination of pH value stability: β-1,3-1,4-glucanase was used respectively with disodium hyd...
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