Genetic engineering inulase and method for preparing crystalline fructose with jerusalem artichoke as raw material
A technology of genetically engineered bacteria and inulinase, which is applied in the field of genetic engineering of enzymes, can solve the problems of low yield of crystalline fructose, inability to effectively utilize glucose components and fructose mother liquor, and high production costs, so as to save economic costs, Improve efficiency and reduce operational difficulty
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0044] Example 1: Individual Expression of Inulinase I and Inulinase II
[0045] (1) Individual expression of inulinase I
[0046] Construction and identification of expression vector: Aspergillus niger was streaked and activated on PDA solid medium, and cultured for 5 days. The spores were picked and inoculated into YPD medium, cultured on a shaker at 28°C for 20-24 hours, and the bacteria were collected. The total RNA of Aspergillus niger was extracted with TRNzol total RNA extraction reagent. Using total RNA as a template, referring to the instructions of the RT-PCR kit, using oligo(dT) as a primer to synthesize the first-strand cDNA by reverse transcription, and then respectively using the first-strand cDNA as a template, using primers F1 (SEQ ID NO: 3) and R1 ( SEQ ID NO: 4) perform PCR to amplify the gene of inulinase I (ie, the inulinase gene inuI, the nucleotide sequence of which is shown in SEQ ID NO: 1). The PCR reaction conditions were as follows: pre-denaturatio...
Embodiment 2
[0050] Example 2: Tandem Expression of Inulinase I and Inulinase II
[0051] respectively according to figure 1 The recombinant bacteria expressing inulinase I and inulinase II in tandem are constructed in the three ways shown. The specific construction method is as follows:
[0052] (1) Construction of recombinant bacteria preferentially expressing inulinase I.
[0053] according to figure 1 In the manner of a, the recombinant plasmid pPIC9K-inuII was used as a template, and F3 (SEQ ID NO: 7) and R3 (SEQ ID NO: 8) were used as primers for PCR amplification. The amplified DNA fragment was double-digested and purified with AvrII and NotI, and then ligated with the recombinant plasmid pPIC9K-inuI that had undergone corresponding digestion, and transformed into Escherichia coli JM109 competent cells to construct the recombinant plasmid pPIC9K-inuI-inuII. Recombinant plasmid pPIC9K-inuI-inuII was single-digested and linearized with SalI, and the digested product was purified a...
Embodiment 3
[0058] Embodiment 3: Five kinds of recombinant bacteria ferment and produce recombinant enzyme under shake flask condition
[0059] Pick five enzyme-producing strains of recombinant bacteria P.pastorisGS115 / pPIC9K-inuI, P.pastorisGS115 / pPIC9K-inuII, P.pastorisGS115 / pPIC9K-inuI-inuII, P.pastorisGS115 / pPIC9K-inuII-inuI, P.pastorisGS115 A single colony of / pPIC9K-inuI / inuII was inoculated into 50 mL of YPD liquid medium containing 50 μg / mL kanamycin and cultured overnight. Insert 1% to 5% of the inoculum into 50mL BMGY liquid medium and cultivate to OD 600 = 2-6 (about 16-18 hours), and then inoculated with the same inoculum amount in 1 / 5-1 / 10 volume of BMMY medium to induce expression, specifically refer to the operation manual provided by Invitrogen. Simultaneously inoculated yeast strain transformed with empty vector was used as control. Shake flask culture conditions were 30°C, 200r / min, 0.5% methanol was added every 24 hours, and samples were taken for enzyme activity dete...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More