Expression Of Protein-glutaminase In Bacillus Subtilis

Protein glutaminase?EC 3.5.1.44,abbreviated as PG?is a novel protein deaminase without protease activity and transglutaminase activity.It can hydrolyze the side chain amido groups of protein-bound or some small peptides-bound glutaminyl residues to generate ammonia and protein-L-glutamic acid.Deamidation of proteins can improve protein functionalities such as solubility,emulsification,and foaming and gelation properties,which are desired properties in some food proteins.PG has great application prospect and broad exploration space in food industry.Because the method of mutagenesis to increase PG yield has no directionality,long process and huge workload,it is necessary to develop an engineering bacterium that can express PG efficiently.Bacillus subtilis expression system is an excellent choice for producing heterologous protein.However,previous research showed PG was unsuccessfully expressed in Bacillus subtilis and its activity was very low.Therefore,the main purpose of this paper is to realize the effective expression of protein glutaminase in Bacillus subtilis,to explore some properties of PG produced by Bacillus subtilis,and to make a contribution to the high expression of PG in Bacillus subtilis in the future.The main contents of this paper are as follows:1.Protein-glutaminase was expressed as a prepro-protein with a putative signal peptide of 21 amino acids and a prosequence of 114 amino acids.In order to make PG more suitable for expression in Bacillus subtilis,its codons were optimized,and two types of secretion expression vectors with different expression formats were constructed:Two mature peptide expression vectors pHT43-mpg,pCW-mpg and a propeptide expression vector pHT43-pp were all cloned into Bacillus subtilis WB800N.However,in the Bacillus subtilis expression system,only pHT43-pp can effectively express the target protein in the pro-PG form,and the mature peptide expression vector cannot successfully express mPG.2.Pro-PG has no activity unless its pro is removed.In order to obtain active PG from pro-PG,we made further research on the digestion of pro-PG by tripsin,alkaline and neutral protease,respectively.It was confirmed that the enzymatic activity can be generated by trypsin digesting pro-PG.The enzyme activity could reach 1.048 U/mL when using 0.15-0.60mg/mL of trypsin with digestion of pro-PG for 0.5-3 h.Then,the digestion effect of alkaline protease and neutral protease to pro-PG expressed by recombinant Bacillus subtilis were studied as well.Fermentation supernatant protein solution containing pro-PG approximately showed 0.75 U/mL of enzyme activity after adding 0.30 mg/mL of alkaline protease and 0.30mg/mL of neutral protease with digestion of pro-PG for 0.5-3 h,respectively.3.In order to get the self-processing active PG from pro-PG in Bacillus subtilis,the recombinant plasmid pHT43-pp was transformed into Bacillus subtilis 168 and DB403 which can produce abundant proteases.The recombinant strain pHT43-pp/168 and pHT43-pp/DB403 have both showed the ability to process pro-PG to active PG through digestion of the proteases generated from themselves.About 0.7 U/mL of enzyme activity was obtained in the fermentation broth after IPTG induction for about 28-32 h.4.In order to investigate the enzymatic properties of PG produced by Bacillus subtilis,pro-PGs with His-tag were expressed.The mPG-His from pro-PG-His digested by trypsin was then purified by nickel column.The purified mPG-His was revealed to be a 20 kD protein and the specific activity reached 19.003 U/mg.And purified mPG-His was used to analyze the properties.The mPG-His sample has good pH adaptability and stability,good temperature adaptability and poor temperature stability.Vmax of mPG-His was 1.381?M?NH₃??min^-1?mL^-11 and Km was 2.983 mM when Cbz-Gln-Gly was using as an substrate,and Vmax of mPG-His was 0.637?M?NH₃??min^-1?mL^-1 and Km was 4.593 mM when casein was an substrate.A variety of protein substrates tested such as soy protein,wheat protein,skim milk,etc,can all be deamidated by mPG-His.The effect of metal ions on enzyme activity is obvious.Cu²⁺,Cu²⁺,Fe³⁺and Fe²⁺have obvious inhibitory effect on enzyme activity.NaCl⁺and Li⁺have inferior stabilizing effect on enzyme activity.Co²⁺has a good stabilizing effect on enzyme activity.Three kinds of metal salts,Mg²⁺,Ca²⁺,Zn²⁺,can stabilize even active the activity of mPG-His.In summary,the expression of protein glutaminase in Bacillus subtilis was successful,the approach of pro-PG processing by an exogenous protease was explored,and the self-processing active PG from pro-PG in Bacillus subtilis was solved.The purified mPG-His sample was used to explore the properties of PG produced by Bacillus subtilis.Studies in this article make a contribution to the better application of PG in the food industry.