Studies On Expression Of Acid Phosphatase By Pichia Pastoris And Its Preliminary Activity

Acid phosphatase is an enzyme that catalyzes the hydrolysis of various phosphate monoesters under acidic conditions.It removes the phosphate group of the substrate molecule by hydrolyzing phosphate monoesters and generates phosphate ions and free hydroxyl groups.Acid phosphatase is widely distributed in animals,plants and microorganisms.Its enzymatic activity is the basic step for cells to play a functional role.It is known that it plays an important role in the efficient use of phosphorus in soil by plants,clinical diagnosis and prognosis of malignant tumors,targeted preparations for cancer treatment and the use of immobilization technology to make biosensors.As a non-specific phosphate-inhibiting acid phosphatase,Morganella acid phosphatase has a wide range of substrate non-specificity and can catalyze the hydrolysis of various phosphate monoesters.Compared with other acid phosphatases,M.morganii acid phosphatase does not require metal ion cofactors and is not easily inhibited by phosphate monoesters when it plays an enzymatic role,which can further provide a new idea for studying the role and function of acid phosphatase.At present,there are many studies on the isolation and extraction of acid phosphatase from various animals and plants and the expression of acid phosphatase in Escherichia coli expression system,but there are few studies on the expression of acid phosphatase in Morganella morganii by Pichia pastoris system.In addition,compared with the above preparation methods,the Pichia pastoris system has the characteristics of high efficiency,easy induction of expression and suitable for high-density culture.Therefore,this study attempts to use the Pichia pastoris expression system to express it.Two Pichia pastoris expression systems,p PICZαA-APT and p GAPZαMUT-APT,were designed and constructed to express the acid phosphatase of Morganella.Among them,the GAPZαMUT vector is a recombinant vector p GAPZαA-6His-Asn-Gly constructed by the expression vector p GAPZαA and the fusion tag-6His-Asn-Gly,which can facilitate purification and increase protein expression yield.Then,PIC-APT4 was selected as the subsequent engineering bacteria for protein purification,optimization of culture conditions,pilot fermentation and preliminary exploration of enzymatic properties.The specific research contents are as follows:1.Construction of Pichia pastoris expression system of acid phosphataseIn this study,the wild-type Morganella acid phosphatase gene was used as the starting sequence,and the codon preference of Pichia pastoris was optimized.The restriction sites Xho I and Not I,Xho I and Eco R I were added to both ends of the acid phosphatase gene sequence to construct p PICZαA/p GAPZαMUT-APT expression vectors.The results of double enzyme digestion showed that the specific band at 816 bp was consistent with the target gene fragment,which proved that the recombinant plasmid was successfully constructed.The recombinant plasmid was linearized and introduced into Pichia pastoris competent cells.The specific band at30k Da was verified by bleomycin resistance screening and protein gel electrophoresis,which was consistent with the expectation.The protein was purified by affinity chromatography,the protein content was determined by BCA method and the enzyme activity was determined by p-nitrophenyl phosphate disodium method.The results showed that the protein content of PIC-APT4 was 61.2 mg/L and the enzyme activity was 38.5 U/m L.The protein content of GAP-APT2 was 41.3 mg/L,and the enzyme activity was 31.9 U/m L.The protein content of GAP-APT3 was 45.8 mg/L and the enzyme activity was 34.3 U/m L.After gene sequencing of PIC-APT4,it was finally selected as the engineering bacteria.2.Optimization of PIC-APT4 culture conditionsThe effects of carbon source concentration,inducer concentration,p H and temperature on protein expression were studied by single factor experiment and Box-Behnken experiment.The results of single factor optimization showed that the best carbon source for protein expression was glucose,the carbon source concentration was 2%,the nitrogen source was peptone,the nitrogen source concentration was 2%,the inducer concentration was 1.5%,the p H was 6.5,the temperature was 28℃,the inoculation amount was 2%,and the fermentation time was96h.On this basis,Box-Behnken design four-factor three-level experiment was carried out.The independent variables were nitrogen source concentration,inducer concentration,temperature and p H,and the dependent variable was protein yield.The regression model was used to analyze and predict the data.The theoretical optimization process conditions were as follows:carbon source concentration 2.21%,inducer concentration 1.34%,p H 6.49,temperature 28.06℃.The protein expression level under this condition was 73.3 mg/L.3.PIC-APT4 pilot fermentationOn the basis of the optimization experiment,the pilot fermentation of 50L fermentor was carried out.The optimum time for inoculation of primary seed solution and secondary seed solution was analyzed.The results showed that the inoculation time was 16h and 18h,respectively.The fermentation was divided into three stages:glucose batch fermentation,glucose fed-batch culture and methanol fed-batch induction.The cell growth stage was supplemented with glucose feeding medium for6h,and the protein expression stage was supplemented with methanol for induction.The whole fermentation process was co-cultured for 96h.The fermentation broth was treated by crude separation,ultrafiltration,purification and dialysis concentration.The protein gel electrophoresis showed that the protein had a specific band at 30k Da,the yield was 217.5 mg/L,and the enzyme activity was 133.6 U/m L,indicating that a relatively pure target protein product was obtained.4.Preliminary study on enzymatic propertiesIn this study,the enzymatic properties of acid phosphatase were preliminarily explored by p-nitrophenyl phosphate disodium method.The results showed that the optimum p H of acid phosphatase was 5.5,and it could not exist stably in acid and alkali environment.The optimum temperature is 35℃and is sensitive to temperature.High temperature will lead to a significant decrease in enzyme activity.The presence of Ca²⁺in the reaction system has a strong inhibitory effect on the activity,the presence of Mg²⁺and Zn²⁺has no obvious promoting effect on the activity,and the presence of Co²⁺has a strong promoting effect on the activity.In summary,this study successfully constructed p PICZαA-APT and p GAPZαMUT-APT expression vectors and screened engineering bacteria.By optimizing the expression of PIC-APT4 engineering bacteria and pilot fermentation,the expression of acid phosphatase was increased.In addition,the enzymatic properties of acid phosphatase were preliminarily explored,which laid a foundation for the subsequent enzymatic research of acid phosphatase.