Trichoderma harzianum with biotransformation characteristics and application thereof
A technology for transforming characteristics and new organisms, applied in the fields of biotechnology and medicine of natural products, can solve problems such as low efficiency, poor product quality, and long cycle, and achieve the effects of short fermentation time, reduced occurrence, and high conversion rate
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Embodiment 1
[0015] Example 1. Trichoderma harzianum transformed geniposide from Gardenia jasminoides to prepare genipin
[0016] Inoculate the Trichoderma harzianum liquid on the PDA slant medium, culture it in a constant temperature incubator at 28-30°C for 3-5 days, dissolve the spores on the slant with sterile water to obtain the spore liquid, and insert the spore liquid into a 4% Gardenia The seed culture medium of powder, seed culture medium is tap water configuration, and pH is natural, and the content of geniposide in the gardenia powder is 6.4% (HPLC measures), after seed culture 24h, access fermentation medium, fermentation medium is to contain 8 1 / 15 mol / L phosphate buffer solution of % gardenia powder, pH 6.1, inoculum size 4%. The culture temperature is 30°C, the rotational speed is 150 rpm, and the fermentation time is 48 hours. After the fermentation, the content of genipin is determined by HPLC method. The conversion rate is calculated as: conversion rate (%)=(moles of geni...
Embodiment 2
[0019] Example 2. Separation and purification of geniposide-β-glucosidase from Trichoderma harzianum
[0020] Take 1L gardenia fermented liquid, centrifuge at 12000rpm, 4°C for 30min to obtain supernatant, precipitate with 75% ammonium sulfate, let stand overnight, centrifuge at 12000rpm, 4°C for 20min, obtain protein precipitate, and use 40 mL pH5.0, 66.7 mmol Dissolve in / L phosphate buffer solution, concentrate by ultrafiltration, pass through DEAE anion column chromatography, DEAE is used for decolorization, the enzyme activity part flows through directly, collect the flow-through liquid, obtain crude enzyme solution, concentrate by ultrafiltration, pass through Superdex200 gel column layer Analysis to obtain pure enzyme solution, SDS-PAGE electrophoresis showed a single band. The molecular weight of the enzyme was determined to be 74.4 kDa by MALDI / TOF mass spectrometer.
Embodiment 3
[0021] Example 3. Geniposide-β-glucosidase converts geniposide to prepare genipin
[0022]Dissolve geniposide in pH 5.0, 66.7 mmol / L phosphate buffer to prepare 1 mg / mL geniposide standard solution, take 200 μL of the solution, preheat at 50°C for 5 minutes, then add Prepare 200μL of pure enzyme solution, react for 1h, 1.5h, 2h, 2.5h, 3h respectively, and bathe in boiling water for 5min to stop the reaction. The conversion rate of geniposide was analyzed by HPLC, and each reaction was repeated three times. The results showed that the conversion rate of geniposide was above 90% after 1 hour of reaction, over 98% after 2 hours of reaction, and no discoloration of the reaction solution and no formation of gardenia blue pigment after 3 hours of reaction.
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