Zymoprotein with beta-galactosidase activity and applications thereof
A technology of galactosidase and enzyme protein, which is applied in the field of enzyme protein with β-galactosidase activity, can solve the problem of inability to hydrolyze agar oligosaccharides, and achieve mild conditions, high catalytic activity, and environmental friendliness
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Embodiment 1
[0033] Example 1: Determination of the gene fragment of β-galactosidase AgWH2A
[0034] In the previous study, Du Zongjun's laboratory screened a high-yielding agarase strain A.gilvusWH0801, which had cloned and expressed three β-agarases AgWH50A, AgWH50B and AgWH50C, and a new agarobiohydrolase AgaWH117, based on the published β-galactosidase used in agarose degradation, four β-galactosidase genes and their gene sequences were obtained by sequencing the whole genome of A.gilvus WH0801, labeled agWH2A, agWH2B, agWH2C and agWH2D. The inventors of the present application analyzed through the phylogenetic tree ( figure 1 ), the agWH2A fragment encoding gene belongs to the glycoside hydrolase GH2 family, the amino acid sequence of its encoded protein is SEQ ID NO: 1, and the nucleotide sequence of the encoded gene is SEQ ID NO: 2; NCBI comparison results show that the agWH2A enzyme of the present invention is compatible with β -galactosidase protein (No. SJN27606) from Vibrio sp....
Embodiment 2
[0035] Example 2: Cloning, expression and purification of β-galactosidase AgWH2A
[0036] Agarophage WH0801 was cultured in 2216E seawater medium, and the DNA genome was extracted with a kit. According to the determined agWH2A gene, design the full-length primer of the gene, the upstream primer: ACGC GTC GAC ATGCCTTGTAAAACTTTA (SalⅠ restriction site), downstream primer: AAATAT GCGGCCGC GCCATGGTTCACCTCTAG (NotⅠ restriction site). Using the genome as a template, the conditions are: 98°C for 2 minutes, then 98°C for 10 seconds, 55°C for 15 seconds, 72°C for 15 seconds, 3 steps for 35 cycles, and finally 72°C for 4 minutes. Agarose gel electrophoresis showed that there was an obvious single band at the position of 2.0kb, and the single band was cut and recovered from the gel, which was the fragment of the agWH2A gene. Digested with SalⅠ and NotⅠ, the target fragment after digestion was ligated with the pET-21a(+) vector that was digested with the same restriction enzyme, the ...
Embodiment 3
[0038] Example 3: Research on the enzymatic properties of β-galactosidase AgWH2A
[0039] (1) Optimum temperature and temperature stability
[0040] The process uses o-nitrophenyl β-D-galactopyranoside (oNPG) as a substrate, in 50mM PBS buffer solution of pH 8.0, detects 10°C, 20°C, 30°C, 35°C, 40°C, 45°C ℃, 50℃, 60℃ effect on enzyme activity. image 3 a shows that the relative activity of AgWH2A reaches the highest at 40°C, and it still maintains 75% activity in the range of 30°C-45°C. When the temperature is higher than 50°C, the enzyme activity will decrease rapidly. image 3 c shows that when AgWH2A is incubated at 30°C and 35°C for 12 hours, the enzyme activity can still maintain more than 80% of the activity, but when it is placed at 40°C, after 5 hours, only 30% of the activity remains. When the temperature rose to 50°C, the enzyme activity dropped sharply after 30 minutes. Based on the results, the optimum temperature and temperature stability of the recombinant β-g...
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