A kind of modified agarase and its application
An agarase and new agar oligosaccharide technology, which is applied in the field of enzyme engineering, can solve the problems such as the inability to obtain a large amount of new agar oligosaccharides with a low degree of polymerization, and achieves the effects of convenient control, optimized conditions and low control accuracy.
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Embodiment 1
[0034]Example 1: Modification and recombinant expression of agarase gene
[0035]Perform bioinformatics analysis and function prediction of existing agarase genes. The structure of the original agarase gene was predicted by NCBI-BLAST, and the results showed that there was a homologous sequence of the Porsecretion system C-terminal sorting domain in the corresponding amino acid sequence at the 5'end of the gene. Through PCR technology and primer design, the nucleotide sequence corresponding to the domain at the 3'end of the existing agarase AgaM1 gene was truncated to obtain the artificial truncated agarase gene AgaM1-01 (SEQ ID NO: 2 ). Connect AgaM1-01 into the expression vector pEASY-Blunt E1 to obtain the recombinant vector pEASY-AgaM1-01, and transfer the recombinant vector into E. coli BL21(DE3) strain;
[0036]The recombinant E. coli strain was inserted into LB liquid medium and cultured at 37°C until the OD 600nm was about 0.5, and isopropylthiogalactoside (IPTG) was added to a fi...
Embodiment 2
[0037]Example 2: Optimization of the production conditions of New Agar Oligo
[0038]Optimization of agar concentration. In theory, the higher the agar concentration, the higher the yield of oligosaccharides. However, as the reaction progresses, the enzyme activity begins to decrease, eventually leading to part of the agar not participating in the reaction, and this phenomenon occurs mostly in high-concentration agar. After the reaction, the unreacted agar solidifies into solids as the temperature decreases. These colloidal solids can block the pipes and filter membranes of production equipment, resulting in waste of substrates, equipment losses and additional labor costs. Therefore, in the production process, substrate utilization is the first optimization factor to consider. The enzyme activity was determined by the dinitrosalicylic acid method, and the substrate utilization rate of 0.5%, 1.0%, 1.5% and 2.0% agar concentration was determined (substrate utilization rate = (initial rea...
Embodiment 3
[0041]Example 3: Composition analysis of new agar oligosaccharide products
[0042]The degradation products and the degree of polymerization of the original agarase (AgaM1) and the truncated agarase (AgaM1-01) used herein were determined by thin layer chromatography (TLC). It was found that the original agarase AgaM1 can only produce new agar oligosaccharides (new agarose and new agarose) with a low degree of polymerization (Figure 3A ), and the truncated agarase (AgaM1-01) can obtain a new agar oligosaccharide with a degree of polymerization of 4-12 under the above optimal conditions (Figure 3B). The above results indicate: 1) Through artificial modification, the original agarase obtained new enzymatic properties after artificial truncation; 2) The above-mentioned optimal production conditions are effective, and the truncated agarase (AgaM1-01) Under the above optimal conditions, new agar oligosaccharides with a degree of polymerization of 4-12 can be obtained, and these oligosacchari...
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