A kind of galactosidase and polynucleotide encoding this enzyme
A galactosidase and polynucleotide technology, applied in the field of β-galactosidase, can solve the problems of indigestion of lactose and affect the growth of suckling piglets, so as to improve the efficiency of feed utilization, treat the diarrhea of suckling piglets, reduce the Effects of production of toxins and enzymes
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Embodiment 1
[0019] Example 1: Cloning and expression of β-galactosidase
[0020] 1. Cloning and expression of galactosidase
[0021] 1. Sample metagenomic DNA extraction
[0022] (1) Sample pretreatment
[0023] Fully oscillate the muddy water sample of 65℃ hot spring, the muddy water is evenly mixed, take 30ml sample and centrifuge at 500g for 5min, let most of the sediment in the sample settle, remove the precipitated sand, centrifuge the remaining solution at 1000g for 10min again, discard The purpose of precipitation is to further remove the relatively small impurities carried in the sample. Finally, the suspension containing the bacteria is centrifuged at 5000g for 10 minutes, the supernatant is discarded and the precipitated bacteria are collected.
[0024] (2) Metagenomic DNA extraction: Qiaampdnaminikit kit was used to extract DNA, and the method was carried out according to the kit instructions;
[0025] ① Suspend the bacteria with an appropriate amount of TEbuffer (20mMTris-H...
Embodiment 2
[0080] Embodiment 2: the activity analysis method of galactosidase and some enzymatic properties
[0081] 1. Exploration of the enzymatic properties and enzyme activity of galactosidase (GAL)
[0082] Dilute the substrate o-nitrophenyl β-D-galactopyranoside (ONPG) with phosphate buffer (NaCl8g, KCl0.2g, NaCl 2 HPO 4 1.42g, KH 2 PO 4 0.27g, dilute to 1L, pH7.4) to make a solution with a concentration of 20mmol, take 10μl of enzyme solution and add 2ml of ONPG solution, take it out immediately after 30min in water bath at 37℃, put it in ice water to cool, and add 4ml of 0.5M / L Na at the same time 2 CO 3 solution to terminate the reaction. The blank sample is to add 10 μl of high-temperature inactivated crude enzyme solution to 2ml ONPG solution, take it out immediately after 30 minutes of water bath at 37°C and place it in ice water to cool, and add 4ml of 0.5M / L Na at the same time 2 CO 3 Solution, measure the absorbance at 405nm.
[0083] 2. Optimum reaction temperatur...
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