Enzymatic Analysis Of Acid Protease From Germinated Chenopodium Quinoa Willd And Optimization Of Production Conditions

Chenopodium quinoa Willd is a kind of food that can meet the basic nutritional needs of human body by monomer plants.It is recommended by FAO as the most suitable nutritional food for human beings.Quinoa is not only rich in a variety of nutrients needed by humans,but also a rare plant with amino acid composition close to human body.Quinoa is widely used in food,feed and agriculture.Phytogenic proteases are widely used in health food,cosmetics,medicine,daily necessities and other fields because of their natural harmlessness and high catalytic activity.Quinoa possesses the complete protein only possessed by animals,and its protein components are very rare in plants,so its protease has great potential for development and research value.In this study,the acid-base properties,separation and purification methods,enzymatic properties,extraction technology and culture conditions of protease in germinated quinoa were studied.The main research results of this paper are as follows:（1）To investigate the effect of different extracting solvents on enzyme activity.In this experiment,deionized water,Tris-HCl buffer and phosphate buffer used to extract germinated quinoa protease.For germinated quinoa protease,phosphate buffer was the most effective,while borate buffer was the worst.On this basis,the protease activity of germinated quinoa protease was detected by using different acid and alkaline substrates.The results showed that the activity of germinated quinoa protease was the highest under the reaction condition of pH 3,and it was an acid protease.The purifying effects of（NH₄）₂SO₄ salting-out method and isoelectric point precipitation method on protease in germinated quinoa were compared.The recovery rates of protease activity from germinated quinoa by（NH₄）₂SO₄ salting-out method and isoelectric point precipitation method were 40.36%and 45.3%,respectively.The purifying times were 2.26 and 2.67 times,and the specific activities were4.63U/mg and 5.46 U/mg,respectively.Therefore,the isoelectric point precipitation method was selected to purify germinated quinoa protease from quinoa extract.（2）On the basis of purifying germinated quinoa protease crude enzymes,the crude enzymes were purified by HiPrep Q XL 16/10 anion exchange column.The purifying effect was ideal.The specific activity of the enzymes was increased to 24.74U/mg with a recovery of 15.65%and a purification multiple of 12.07.The electrophoretic analysis of the purified protease showed that the purified enzyme showed a single band and the molecular weight of the germinated quinoa protease was about 55 kDa.（3）The results of enzymatic properties of germinated quinoa protease showed that the optimum pH of the enzyme was 3 and the optimum temperature was 60℃.The temperature stability and pH stability of the enzyme were not strong.As for metal ions,Cu²⁺and K⁺had slight activation on the protease,Mn²⁺had the strongest activation,while Zn²⁺and Fe²⁺had inhibitory effect on the activity of the protease.The results of protease inhibitors showed that aspartate protease inhibitors basically inhibited the activity of acid protease from germinated quinoa,so it is very likely that acid protease from germinated quinoa is an aspartate protease.The kinetics of enzymatic reaction showed that when casein was used as substrate,Vmax=0.285mg/min,Km=3.52 mM.（4）In this experiment,germinated quinoa was used as raw material and phosphate buffer as extracting solvent.Furthermore,single factor experiments were carried out on the extraction pH,the ratio of material to liquid and the extraction time.In order to make the experimental results more scientific and accurate,orthogonal experiments with three factors and three levels were carried out.The activity of germinated quinoa protease was taken as an index to optimize the extraction parameters of germinated quinoa.The optimum extraction parameters of germinated quinoa protease were obtained as follows:the germinated quinoa was fully homogenized in a buffer of pH 7.0 with the ratio of material to liquid of 1:6,and then extracted at 4℃for 30 minutes.（5）On this basis,taking the activity of germinating quinoa proteinase as an index,the single factor experiments of germination time,temperature and relative humidity of quinoa were carried out.In order to make the experimental results more scientific and accurate,orthogonal experiments with three factors and three levels were carried out.Taking the activity of quinoa protease as an index,the germination conditions of quinoa were optimized,that is,quinoa acid protease activity produced by quinoa seeds germinated at 25℃and 60%relative humidity for 18 h was the highest.（6）By means of bioinformatics,the molecular weight of quinoa aspartate protease was predicted to be 54.993 kDa,the theoretical isoelectric point was 4.56,the liposolubility parameter was 84.80,and the average coefficient of hydrophilicity was 0.002.The glycosylation site analysis showed that there was no N-glycosylation site in quinoa aspartate protease.The location of the enzyme in quinoa（vacuoles）was predicted.Two enzyme activity sites Asp94 and Asp277 were predicted,and its 3D structure was predicted.The plasmid carrying quinoa aspartate protease was successfully transferred into the host of E.coli,and the reason why quinoa aspartate protease could not be expressed was analyzed.