Hybrid enzyme S6PE-A containing pectinesterase S6PE-PE and polygalacturonase S6PE-PG, and gene and application of hybrid enzyme S6PE-A
A technology of S6PE-PE and S6PE-PG, applied in the field of genetic engineering, can solve problems such as decreased enzyme activity
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Embodiment 1
[0061] Example 1 Cloning of the double-domain hybrid enzyme encoding gene S6PE-A derived from Penicillium sp.Sx6
[0062] Extract genomic DNA and RNA. The degenerate primers PE8-F and PE8-R were designed and synthesized according to the conserved sequence [F(KT)G(YF)QDT and LGRPW(RG)(DN)] of the pectin esterase fungal gene of the eighth family of sugar esterase:
[0063] PE8-F: 5'-TTCAMNGGNTWYCARGAYAC-3';
[0064] PE8-R: 5'-GTCGCBCCAGGGBCGICCIAR-3'
[0065] PCR amplification was performed using the above-mentioned Penicillium sp.Sx6 genomic DNA as a template. The parameters of the landing PCR reaction are: denaturation at 94°C for 5min; denaturation at 94°C for 30sec, annealing at 55-50°C for 30sec, extension at 72°C for 1min, 10 cycles (0.5°C for each cycle), denaturation at 94°C for 30sec, annealing at 50°C for 30sec , 72°C for 1min, 30 cycles, 72°C for 10min. A fragment of about 280bp was obtained, which was recovered and connected with the pEASY-T3 vector and sent to S...
Embodiment 2
[0071] Example 2 Preparation of recombinant enzyme protein.
[0072] The expression vector pPIC9 was subjected to double digestion (SnaBI+NotI), and the gene S6PE-PE encoding pectin esterase, the gene S6PE-PG of polygalacturonase and the gene S6PE- A Double enzyme digestion (SnaBI+NotI), the cut gene fragments encoding mature pectinesterase, polygalacturonase and double domain hybrid enzyme (removing the signal peptide fragment) were connected with the expression vector pPIC9 to obtain Recombinant plasmids pPIC9-S6PE-PE, pPIC9-S6PE-PG and pPIC9-S6PE containing pectin esterase gene S6PE-PE, polygalacturonase gene S6PE-PG and double domain hybrid enzyme gene S6PE-A -A. And transform Pichia pastoris GS115 to obtain recombinant yeast strains GS115 / S6PE-PE, GS115 / S6PE-PG and GS115 / S6PE-A.
[0073] At the same time, the above method was used to construct the expression vector of the gene fragment of the double-domain hybrid enzyme without removing the signal peptide, and transform...
Embodiment 3
[0077] Activity Analysis of Embodiment 3 Recombinase Protein
[0078] 1. Detection method
[0079] Polygalacturonase activity was determined by DNS method. The specific method is as follows: under the given pH and temperature conditions, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, react for 10 min, add 1.5 mL of DNS to terminate the reaction, and boil for 5 min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose polygalacturonic acid to generate 1 μmoLD-(+)-galacturonic acid per minute under given conditions.
[0080] The activity of pectin esterase was determined by constant pH titration. The specific method is as follows: under the given pH and temperature conditions, the reaction system includes 25mL substrate (adding 0.117M NaCl) and 1mL appropriately diluted enzyme solution, and react for 10min. According to the amount of carb...
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