Research On Molecular Modification And Expression Of Protein Glutaminase And Its Application

Protein glutaminase（EC 3.5.1.44,PG）is a deamidase that has been widely concerned in recent years.It can specifically remove the amide group of glutamine from the side chain of protein,improving the solubility and functional properties of plant-based proteins.It has potential applications in the food industry.However,PG is limited by its low enzymatic properties,lack of studies on enzyme production level and functional properties.Therefore,it is of great significance to improve the catalytic performance and heterologous expression level of PG.In this study,the safe and efficient expression of PG in Bacillus subtilis 168 was achieved through the semi-rational design of PG molecular modification and the construction of D-Alanine defected expression system.In addition,the structure improvement of wheat gluten（WG）by PG deamidation and its application in the construction of high internal phase emulsions were evaluated to provide guidance for the functional improvement of plant-based proteins.The main results are as follows:（1）Molecular modification of PG based on semi-rational design strategy to improve the catalytic activity.The software Discovery Studio was used to conduct molecular simulation docking between PG and substrate,and 9 key amino acid sites potentially affecting the binding of PG and substrate were selected to construct a saturated mutant library to screen highly active PG mutants.The enzyme activity of the optimal PG mutant A291S reached 5.10U·m L^-1 at shaking flask level,which increased by 45.71%compared with initial PG.The K_catand catalytic efficiency(K_cat/K_m)were increased by 30.40%and 27.66%respectively.Further molecular dynamics（MD）simulations of the initial PG and the mutant A291S showed that the mutation introduced more hydrogen bonding,which may increase substrate flux to and from the pocket and thus increase enzyme activity.（2）The D-Alanine deficient Bacillus subtilis system was constructed to promote the safe and stable expression of PG.In order to avoid the addition of antibiotics during fermentation,a D-Alanine defective expression system was constructed in Bacillus subtilis 168 by using the Cre/lox recombinant system to knock out the alanine racemase coding gene（dal）.The study on the expression stability of the strain showed that the D-Alanine deficient system exhibited higher stability than the antibiotic system.The enzyme activity of mutant A291S was 5.25U·m L^-1,which was similar to that of kanamycin resistant system before modification(5.10U·m L^-1).In the expanded culture of 5-L fermenter,the activity of fermentation enzyme reached 8.15 U·m L^-1.（3）The improvement of WG structural properties by PG and its application in the construction of HIPE were evaluated.In this study,PG was used to deamide WG.The deamidation degree（DD）increased and reached 70.50%after 48 h with the continuous deamidation reaction.The solubility was improved with no significant change in molecular weight.WG with different DD was selected as stabilizer to prepare HIPE.Compared with WG,deamidation WG（DWG）results in stable HIPE.In addition,the HIPE stabilized by DWG with moderate DD（28.70-40.50%）has denser network structure,higher modulus of elasticity and relatively lower coefficient of oral friction.