Heat-resisting phytase Pichia pastoris engineering bacterial strain and production method of heat-resisting phytase

Abstract

The invention provides the construction of a Pichia pastoris engineering bacterial strain GS115 / appA NR for producing heat-resisting recombinant phytase and a method for producing the heat-resisting phytase by using the bacterial strain. A heat-resisting phytase gene is synthesized by adopting the two-step PCR method and a recombinant plasmid is constructed to be transferred into the Pichia pastoris bacterial strain GS115 through electric shock. The invention comprises the following steps of: (1) cloning the heat-resisting phytase gene synthesized by the two-step PCR method into a Pichia pastoris secretive expression vector to obtain a Pichia pastoris recombinant expression plasmid; (2) transferring the ombinant expression plasmid to the Pichia pastoris host GS115 through electric shock, screening by using an MD flat board and a substrate flat board containing calcium phytate, and then screening out a bacterial strain with largest hydrolysis circle; and (3) choosing positive clone and inducing to express by using 0.5% methanol. The heat stability of the recombinant heat-resisting phytase expressed by the Pichia pastoris engineering bacterial strain GS115 / appA NR is much higher than that of E.coli K12 wild type phytase through measurements, and specifically, 92% of enzymatic activity can still be maintained after a water bath at 95 DEG C for 10 min. The fermentation crude enzymatic activity of the recombinant phytase produced by fermenting the Pichia pastoris engineering bacterial strain can reach 978 U / mL.

Description

technical field

[0001] The invention relates to the construction of a heat-resistant phytase-producing Pichia pastoris engineering bacterium and a method for producing heat-resistant phytase by using the strain, belonging to the field of microbial genetic engineering. Background technique

[0002] Phytase has broad application prospects in feed, food and medicine because it can hydrolyze phytic acid and phytate. In terms of animal feed, phytase added to feed can alleviate the anti-nutritional effect of phytic acid and phytate in feed by hydrolyzing phytic acid and phytate. Acidase hydrolysis can reduce the discharge of phosphorus in animal manure, and reduce the pollution caused by phosphorus to the environment. In terms of food industry, although no food-related phytase products have appeared on the market so far, phytase still has great application potential in the food industry. For example, Asians mainly use rice and wheat as their main products. Staple food, and about...

Examples

Embodiment 1

[0021] Example 1: 36 primers (appA)F1-(appA)R36 were designed to amplify the acid-resistant phytase gene by a two-step method. First, the heat-resistant phytase gene with a size of 1299bp was divided into two small fragments of 641bp and 658bp to amplify by PCR, the concentration of the first and last primers was 10 μmol / L, and the concentration of the middle primer was 1 μmol / L, 94 ° C for 20 sec; 60 ℃ for 30sec; 68℃ for 50sec (25cycle); 68℃ for 7min to amplify 641bp and 658bp DNA fragments. Two-step amplification, primer concentration 10 μmol / L, 94°C 20sec; 60°C 30sec; 68°C 1min 30sec (25cycle); 68°C 7min), amplified 1299bp DNA fragment. Insert the above gene into the integrated expression vector pHBM905A of Pichia pastoris, the sequence of the DNA fragment was confirmed to be correct and inserted into the expression vector pHBM905A through sequencing, and then the obtained expression vector containing the heat-resistant phytase gene was introduced into Pichia pastoris In G...

Embodiment 2

[0022] Example 2: The GS115 / appANR strain obtained in Example 1 was cultured in a medium with glycerol as a carbon source in a shake flask at 20-30°C, so that the OD corresponding to the cell density 600 After reaching 20, transfer to the induction medium with methanol as the only carbon source and induce culture at 20-30°C. Induce and cultivate for 216 hours, take samples every 24 hours, and analyze the samples taken. When the enzyme production level reaches more than 978U / mL, stop the induction, centrifuge the bacteria, and collect the supernatant containing heat-resistant phytase, which is Crude enzyme solution, the enzyme activity assay was carried out on the crude enzyme solution, and the enzyme activity was 978U / mL. After 30-75% saturation of ammonium sulfate precipitation and concentration, the high temperature resistant recombinant phytase product can be obtained.

[0023] Use pH 5.5, 0.25M acetic acid buffer to prepare 7.5mmol / L sodium phytate solution, put the above...

technical field

[0001] The invention relates to the construction of a heat-resistant phytase-producing Pichia pastoris engineering bacterium and a method for producing heat-resistant phytase by using the strain, belonging to the field of microbial genetic engineering. Background technique

[0002] Phytase has broad application prospects in feed, food and medicine because it can hydrolyze phytic acid and phytate. In terms of animal feed, phytase added to feed can alleviate the anti-nutritional effect of phytic acid and phytate in feed by hydrolyzing phytic acid and phytate. Acidase hydrolysis can reduce the discharge of phosphorus in animal manure, and reduce the pollution caused by phosphorus to the environment. In terms of food industry, although no food-related phytase products have appeared on the market so far, phytase still has great application potential in the food industry. For example, Asians mainly use rice and wheat as their main products. Staple food, and about...

Examples

Embodiment 1

[0021] Example 1: 36 primers (appA)F1-(appA)R36 were designed to amplify the acid-resistant phytase gene by a two-step method. First, the heat-resistant phytase gene with a size of 1299bp was divided into two small fragments of 641bp and 658bp to amplify by PCR, the concentration of the first and last primers was 10 μmol / L, and the concentration of the middle primer was 1 μmol / L, 94 ° C for 20 sec; 60 ℃ for 30sec; 68℃ for 50sec (25cycle); 68℃ for 7min to amplify 641bp and 658bp DNA fragments. Two-step amplification, primer concentration 10 μmol / L, 94°C 20sec; 60°C 30sec; 68°C 1min 30sec (25cycle); 68°C 7min), amplified 1299bp DNA fragment. Insert the above gene into the integrated expression vector pHBM905A of Pichia pastoris, the sequence of the DNA fragment was confirmed to be correct and inserted into the expression vector pHBM905A through sequencing, and then the obtained expression vector containing the heat-resistant phytase gene was introduced into Pichia pastoris In G...

Embodiment 2

[0022] Example 2: The GS115 / appANR strain obtained in Example 1 was cultured in a medium with glycerol as a carbon source in a shake flask at 20-30°C, so that the OD corresponding to the cell density 600 After reaching 20, transfer to the induction medium with methanol as the only carbon source and induce culture at 20-30°C. Induce and cultivate for 216 hours, take samples every 24 hours, and analyze the samples taken. When the enzyme production level reaches more than 978U / mL, stop the induction, centrifuge the bacteria, and collect the supernatant containing heat-resistant phytase, which is Crude enzyme solution, the enzyme activity assay was carried out on the crude enzyme solution, and the enzyme activity was 978U / mL. After 30-75% saturation of ammonium sulfate precipitation and concentration, the high temperature resistant recombinant phytase product can be obtained.

[0023] Use pH 5.5, 0.25M acetic acid buffer to prepare 7.5mmol / L sodium phytate solution, put the above...