A kind of preparation method of oligogentianose
A technology of oligosaccharide gentisose and glucose, which is applied in the field of fermentation engineering and can solve the problems of low yield and the like
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Embodiment 1
[0040] Example 1: Construction of Pichia pastoris KM71 / pPIC9K-bgl1 genetic engineering bacteria
[0041] According to the known sequence (accession NO.U09580) in Genbank, primers P1 and P2 were designed (the underlined part is the restriction site, respectively SnaB I and Not I), and the Trichoderma viride genome was used as a template for PCR amplification. The DNA fragment bgl1 was obtained. Then use P1 (SEQ ID NO.2), P2 (SEQ ID NO.3), P3 (SEQ ID NO.4), P4 (SEQ ID NO.5) as primers, remove introns by overlapping PCR, and obtain The target DNA fragment bgl1( figure 1 ), then the β-glucosidase gene bgl1 was connected to the yeast expression vector pPIC9K, and the recombinant plasmid pPIC9K-bgl1 was electroporated and integrated into Pichia pastoris KM71. Then spread it on the MD plate, grow single clones on the MD plate, and then screen multi-copy transformants on the YPD-G418 plate, and the G418 concentration screening gradient is 0.5 mg / mL, 1 mg / mL, and 2 mg / mL. Pick a sin...
Embodiment 2
[0047] Embodiment 2: fermentation produces β-glucosidase
[0048] 1. (1) Batch fermentation stage: inoculate the seed liquid in the fermenter with an inoculation amount of 8%-10%, control the temperature at 28-32°C, the initial rotation speed at 180-200rpm, the initial ventilation rate at 7L / min, and the dissolved oxygen at 28 -32%, pH 4.5-5.5; (2) Feed-fed fermentation stage: when dissolved oxygen rises to 80-100%, feed-fed culture is carried out by adding glycerin at a constant rate, controlling temperature 28-30°C, dissolved oxygen 28 -32%, pH 4.5-5.5; (3) induction culture stage: when the bacterial cell concentration reaches OD 600 When the temperature is 80-120, add methanol to induce enzyme production with a methanol feeding device, control the methanol concentration at 0.5-1.5%, control the temperature at 28-32°C, dissolve oxygen at 28-32%, and pH4.5-5.5, and induce 96- 144h. The fermentation broth was centrifuged to take the supernatant to obtain a crude enzyme solut...
Embodiment 3
[0049] Example 3: Enzymatic properties of β-glucosidase
[0050] The obtained β-glucosidase enzyme liquid is characterized enzymatically by the above-mentioned enzyme activity assay method, and the enzyme activity is measured at different temperatures with pNPG as a substrate, and the results show that the optimum temperature of β-glucosidase is at 60°C ( image 3 ). Then set different pH gradients to measure the enzymatic activity of β-glucosidase under optimum temperature conditions, and draw the optimum pH of this enzyme as 5( Figure 4 ).
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