Penicillium oxalicum strain for producing high-temperature polygalacturonase and application of penicillium oxalicum strain
A technology of Penicillium oxalate and polygalactose, applied in the fields of biotechnology and engineering
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Embodiment 1
[0035] Embodiment 1: Isolation and breeding of bacterial strains
[0036] The strain isolation and breeding process includes three steps: sampling, primary screening, secondary screening and purification.
[0037] Sampling: Appropriate soil was collected from citrus orchards in the suburbs of Yangshuo County, Guilin City, Guangxi.
[0038] Primary screening: prepare primary screening medium (pectin 30g, NaNO 3 5g, KCl0.5g, MgSO 4 0.3g, (NH4) 2 SO 4 1.4g, CaCl 2 0.3g, KH 2 PO 4 3.8g, 20g agar powder, 0.2g bromophenol blue, 1000ml water), the initial pH value is 5.5, and sterilized at 121°C for 20min. Steps: Randomly pick about 0.5g sample from the collected soil, suspend it in 10ml sterile water, shake it well, after standing for a period of time, take the supernatant and dilute it to an appropriate multiple, evenly spread it on the primary screening medium, Incubate at 30°C for 2-3 days. Pick colonies with larger yellow hydrolysis circles for further screening.
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Embodiment 2
[0040] Embodiment 2: bacterial strain identification
[0041] The taxonomic identification of the strain PC1 strain screened in Example 1 includes molecular identification and biolog microbial automatic identification system identification.
[0042] Molecular identification process: Inoculate PC1 strain in PDA medium, collect spores after culturing at 30°C for 5-6 days, and press 10 6 The inoculation amount of spores / ml was inoculated in the liquid fermentation medium, cultured on a shaker at 30°C and 200rpm for 48 hours, and the bacterial cells were collected, ground and crushed by freezing with liquid nitrogen, and the genomic DNA of the bacterial cells was extracted by the phenol-chloroform method. Using the DNA as a template, the general primers ITS1 (5-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used for polymerase chain reaction (PCR) to amplify the ITSDNA sequence of the bacterium. The 50ul reaction system contains 5ul of PCR buffer, 2ul each o...
Embodiment 3
[0045] Embodiment 3: strain characteristic experiment
[0046] Strains by 10 6 spore / ml inoculum was inoculated in liquid fermentation medium (containing 1% pectin, 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium dihydrogen phosphate, 0.03% calcium chloride, 0.5% sodium nitrate, 0.1% spit Temperature 80, initial pH value 5.5, sterilized at 121°C for 20min.), 30°C, 200rpm shake flask culture. The enzymatic reaction conditions are: 0.5% polygalacturonic acid substrate, pH 4.8 acetic acid-sodium acetate buffer solution, 50°C for 15 minutes, 1 μg of galacturonic acid residue produced by hydrolyzing the substrate in 1 minute is defined as an enzyme activity unit . The results showed that the polygalacturonase activity in the fermentation broth reached the maximum when the bacteria were cultured for 60 hours, which was about 3445.3u / ml. 36-72h is the stable period of enzyme production, and after 72h, the enzyme production ability is significantly reduced, as sho...
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