A kind of engineering strain of mannosidase and its application
A technology of mannosidase and mannan, applied in the field of bioengineering, can solve the problem of high production cost of mannan, and achieve the effects of easy separation and purification, high product purity and simple operation process
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Embodiment 1
[0038] Example 1: Acquisition of TFMS and expression of recombinant E. coli
[0039] 1. Heterologous expression of TFMS gene
[0040] Taking the TFMS gene sequence in the thermophilic bacteria Thermobifida fusca as a template, the codons were optimized according to the degeneracy of the codons to synthesize a TFMS gene with a size of 1362 bp (nucleotide sequence shown in SEQ ID NO.2), and introduced NdeI and XhoI digestion site, the synthesized product was double digested with NdeI and XhoI, and the digested fragment was purified and recovered. Then, the pET21a empty plasmid was double digested with the same enzyme, purified and recovered, and then ligated with the previously obtained fragment, and the ligated product was transformed into E. coliBL21 (DE3). Take an appropriate amount of transformation solution and spread it on the LB medium plate, cultivate it overnight at 37°C, pick a single colony in shake flasks after the colony PCR is correct, and extract the plasmid. Af...
Embodiment 2
[0043] Example 2 Activity Characterization of Mannosidase TFMS
[0044] Using konjac glucomannan as the substrate, the enzymatic properties of TFMS were characterized.
[0045] 1. Weigh 0.2, 0.4, 0.8, 1.6, 1.8, 2.0, 2.2, 2.4 and 2.8 g of konjac flour, respectively, and dissolve them in 1 L of distilled water to prepare a konjac mannan solution, then add 10 μL of enzyme solution, and react at 37°C for 1 h. Wherein the enzyme liquid is the enzyme powder of the TFMS prepared in Example 1, diluted with deionized water, and the concentration of the dilution solution is 50 μg / mL. like figure 2 As shown in a, the catalytic activity of TFMS increased rapidly as the initial concentration of konjac glucomannan increased from 0.2 g / L to 2.2 g / L. These results indicate that there is no apparent substrate inhibition in fructose biotransformation.
[0046] 2. Determination of optimum temperature: prepare 2g / L konjac glucomannan as the reaction substrate, add 1mL 2g / L konjac glucomannan ...
Embodiment 3
[0053] Embodiment 3 Utilize TFMS enzyme liquid and prepare mannose oligosaccharide product efficiently
[0054] Specific steps are as follows:
[0055] Weigh 0.5 g of konjac flour and dissolve it in 50 ml of distilled water to prepare a konjac mannan solution, then add 5 ml of TFMS enzyme solution, and react at 37°C for 1 h. Wherein the enzyme liquid is the enzyme powder of the TFMS prepared in Example 1, diluted with deionized water, and the concentration of the dilution solution is 50 μg / mL.
[0056] After the sample was processed, gel fluorescence electrophoresis was carried out, and the results were as follows through observation under ultraviolet light. image 3 Shown: With mannose and sucrose as standard controls, the cell disrupting solution added with TFMS can hydrolyze konjac mannan into monosaccharides, disaccharides and trisaccharides, that is, the recombinant expression of thermophilic mannosidase TFMS has a negative effect on the mannan substrate. The compound h...
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