A kind of engineering bacteria for producing high-viscosity xanthan gum and its construction method and application
A construction method, xanthan gum technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve problems such as xanthan gum degradation
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Embodiment 1
[0064] Embodiment 1, be used for the construction of the engineering bacterium of producing high-viscosity xanthan gum
[0065] 1. Construction of overexpression plasmid pBBAD-E
[0066] Using ATCC33913 genomic DNA as a template, the primers E1 and E2 in Table 2 were used for PCR amplification to obtain the gumE gene fragment. Then, the gumE gene fragment was ligated with the pBBAD vector cut with KpnI and PstI using the ClonExpress MultiS One StepCloning Kit, a rapid cloning kit from Nanjing Novozyme Company, to construct the gumE gene overexpression plasmid pBBAD-E. GumE gene overexpression plasmid pBBAD-E expresses GumE protein. The amino acid sequence of GumE protein is shown in SEQ ID No.3.
[0067] The pBBAD-E plasmid was verified by sequencing. Sequencing results show that: the pBBAD-E plasmid replaces the sequence between the KpnI and PstI restriction sites of the pBBAD vector with the gumE gene sequence shown in SEQ ID No.4 (comprising the KpnI and PstI restriction...
Embodiment 2
[0080] Embodiment 2, the application of engineering bacteria for producing high-viscosity xanthan gum in the production of xanthan gum
[0081] 1. Preparation of Xanthan Gum by Fermentation and Culture of Recombinant Bacteria
[0082] 1. Preparation of seed solution
[0083] Pick ATCC33913 and the recombinant strain ATCC33913E obtained in Example 1 to streak on different slant mediums, culture at 28°C for 2 days; then inoculate into seed medium (50mL medium / 500mL shake flask), and cultivate at 28°C at 200rpm to OD 600 =1.2-1.5, take 20-25mL of culture and transfer it to fresh seed medium (500mL medium / 2000mL shake flask), cultivate to OD at 28°C and 200rpm 600 =0.8-1.0, to obtain seed liquids respectively. Gentamicin (final concentration 15 μg / mL) was added to the culture medium of the recombinant strain.
[0084] 2. Fermentation of seed liquid
[0085] 500mL of seed liquid, gentamicin and arabinose were added to 4.5L fermentation medium (Bio Flo 310 fermenter) to obtain ...
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