| Most proteins derived from plant have poor solubility under mild acidic conditions, which are the pH ranges of most food systems. The poor solubility causes the poor functionalities such as emulsification, foaming, oil and water holding capacity of them, resulting in their limited uses in food industry. The main reason was that these proteins from plants generally contain high level of glutaminase residues. Hydrogen bond were easily formed between glutaminase residues and other acylamino groups, which may cause the hydrophilic groups be wrapped inside the protein structure, and hydrophobicity of protein increased, resulting in the poor solubility of proteins. When proteins were deamidated, the interactions between acylamino groups were prohibited, and the hydrophilic groups were exposed after the structure of protein changed. Meanwhile, deamidation cause the increase of negative charges in proteins, which may decrease the pI of protein. These changes lead to an improvement of solubility in acidic conditions, and the problem of poor functionalities was solved.Many studies indicated that enzymatic deamidation has several advantages over chemical deamidation because of its high specificity, mild reaction conditions and safety. Enzymatic deamidation methods using transglutaminase, protease and peptidoglutaminase were studied, and the results showed some side effects of cross-linked or broken of peptide chains during the deamidation. Protein-glutaminase (PG) was a kind of new protein-deamidating enzyme, with well specificity. The enzyme only worked on the glutamine residue of proteins and long peptides, with low side effect of changing other properties of proteins. Therefore, PG have a very good prospect in food industry. This researches focus on the profiles of fermentation process of PG, the purification of PG and the properties and applications of PG. The contents which had been studied in this article as listed below.A PG producing strain Chryseobacterium indologenes, encoding ZYF12O113-1, which was discovered and determined in our laboratory was studied in this research firstly for the fermentation of PG. The result indicated that the culture period reached the plateau after the culture of strain in seed medium for11h. The enzyme activity reached the highest level at the fermentation culture time of12h in flask or10h in fermentation tank, were0.586U/mL and0.405U/mL, respectively. At the same time, an enzyme activity assay method using96-well plate was developed based on the assay method using test-tube, and the accuracy showed no significant different with the assay using test-tube, with good repeatability, which gave some methods for high-throughput screening of strains. Then150suspect PG producing strains were screened by the method of96-well plate and10with higher enzyme producing ability of them were selected and preserved.After knowing the regulation of fermentation of PG, some conditions and factors of fermentation were studied, including temperature, rotate speed, liquid volume in flask, carbon source and nitrogen source. The enzyme activities were determined at the fermentation time of12h, the result indicated that at the optimal conditions of30℃,200r/min,25mL liquid in250mL flask, sucrose as carbon source, and poly-peptone as nitrogen source, enzyme producing of the strains reached the highest level, which was0.612U/mL.The study extracted and purified the PG from crude enzyme solution after fermentation, and finally obtained the PG with high purity. In this article, the process of purification steps, including centrifugation, ultrafiltration, ethanol precipitation, ion exchange and gel filtration were reported in details. After the step of ultrafiltration, most impurities were wiped off and the specific enzyme activity and activity recovery in this step were0.435U/mg and85.37%, respectively. After the step of ethanol precipitation, the specific enzyme activity and activity recovery in this step were2.269U/mg and76.07%. respectively. After the step of ion exchange using SP Sepharose column, PG was further purified, and the specific enzyme activity and activity recovery in this step were8.403U/mg and75.00%, respectively. After the step of gel filtration, the purified PG was finally obtained. The specific enzyme activity was27.371U/mg, the recovery was32.91%, and the purification folds were161.96compared to the original broth supernatant.At the last section of this research, the properties of PG and preliminary applications using soy protein isolate (SPI) as substrate of purified PG obtained were studied. The result showed the optimal pH value and optimal temperature for PG activity were about6-7and60℃, respectively. The enzyme was stable at the environment of pH value of6-8and20-40"C, Among the metal ions and inhibitors, Cu2+showed some inhibition to PG activity, and Na+showed a little improvement to the activity. Other metal ions and inhibitors had no significant effect. By the double-reciprocal Lineweaver-Burk plots, the Km and Vmax of the enzyme were concluded, which were1.68and1.742μg(NH3)×min-1×mL-1, respectively. The result of enzyme specificity indicated that Na-Cbz-Gln-Gly was the best substrate for PG, which was1.983μmol(NH3)×min-1×mg-1; SPI was worse, which was0.41μmol(NH3)×min-1×mg-1. Gelatin was far worse than Na-Cbz-Gln-Gly, which was0.118μmol(NH3)×min-1×mg-1, and bovine serum albumin was the worst, which was0.025umol(NH3)×min-1×mg-1. When the SPI suspensions were treated with PG, it could be proved that SPI was well deamidated after the reaction. At the reaction time of2h, the degree of deamidation (DD) was over40%, and DD reached52.34%at the reaction time of18h.This research studied the fermentation regulations, fermentation conditions, purification of PG and the properties and applications of PG, based on the strain of Chryseobacterium indologenes ZYF120113-1. The result concluded from this research gave some ideas and advices to the researches on PG in the future. |
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