Expression And Enzymatic Properties Of Wheat Catalase And Its Application On The Wheat Flour Processing Quality

Due to the influence of wheat growth environment and post-harvest storage,the quality attributes of wheat flour can’t fully meet the requirements of the flour processing industry in China.Flour improver can improve the processing quality of flour and developing wheat endogenous enzymes as flour improver has become a promising direction in cereal industry.In this paper,wheat catalase（WCAT1）was obtained through prokaryotic and eukaryotic expression systems.Based on its enzymatic properties,the effect of WCAT1 on the processing quality of wheat flour was discussed.The main research contents are as follows:（1）Bioinformatics analysis and Cloning of wcat1 gene.wcat1 was successfully cloned from wheat seedlings.After sequencing,the bioinformatics analysis of wcat1 gene and its encoded protein was performed.Results showed that wcat1 contains 1,479 bp encoding WCAT1 with 493 amino acids.WCAT1 belongs to hydroperoxide-hemagglutinin superfamily,and was a hydrophilic protein with abundant potential N-glycosylation sites.Meanwhile,WCAT1 exhibited a close genetic relationship with CAT from oryza sativa,setaria italica and sorghum bicolor.（2）Recombinant expression of WCAT1.E.coli recombinant expression plasmid and Pichia pastoris eukaryotic expression plasmid were successfully constructed,which achieved the heterologous expression of WCAT1.The intracellular recombinant protein of E.coli system expressed was insoluble inclusion bodies,while the extracellular recombinant protein of Pichia pastoris system expressed possessed biological activity.Choosing the activity of WCAT1 as response value,the recombinant expression conditions of WCAT1 in Pichia pastoris were optimized as follows:the concentration of methanol was 1%（v/v）,inducing expression time was 84 h,medium pH was 6.0,the amount of loaded medium was 10%（v/v）and the initial OD₆₀₀00 was 2.0.In these conditions,the enzyme activity in the fermentation supernatant was 1233±56 U/mL.（3）Purification and enzymatic characteristics of WCAT1.WCAT1 was isolated and purified using ammonium sulfate fractional precipitation,followed by Mono QTM5/50 GL anion exchange column chromatography.The optimum reaction temperature and pH of WCAT1 were 35°C and 7.0 respectively.In addition,WCAT1 presented a superior stability under low temperature and neutral pH.The activity of WCAT1 could be improved by Fe³⁺,but it was inhibited by Zn²⁺,Cu²⁺,Mg²⁺and Fe²⁺.Meanwhile,EDTA,SDS,Tween 20 and tritonX-100 could also significantly inhibit the activity of WCAT1.When hydrogen peroxide was used as the substrate,the K_m and V_max values of WCAT1 were 18.95 mmol/L and 0.22μmol/min,respectively.（4）The effect of WCAT1 on the processing quality of wheat flour.The effects of WCAT1 on flour processing quality was investigated by farinographic,rheological analysis,scanning electron microscopy and texture profile analysis（TPA）.The mechanism of WCAT1 to improve the quality of flour processing was also investigated.Adding WCAT1 with 3000 U/g of flour alone can significantly improve the silty properties of the flour,and increase the dough development time of dough from 2.85 min to 4.15 min.WCAT1 can increase the viscoelastic properties of the dough,increase the specific volume of the bread,and reduce the hardness,gumminess,and chewiness of bread.The reason of WCAT1 improves the processing quality of flour is related to its ability to strengthen the gluten network structure and increase the contents of gluten protein aggregates（GMP）.