Mutagenesis Breeding Of Protein-glutaminase High-producing Strains And Primary Studies On Its Metabolism Regulation

Protein–glutaminase(PG)is a novel deamidating enzyme which hydrolyzes the amino group side chains on protein-bound glutaminyl residues.On one hand,deamidation generally decreases the isoelectric point of the proteins due to the increasing number of the negatively charged carboxyl groups,increases the solubility of the protein,thus PG can be used as a cosolvent in food processing.On the other hand,deamidation leads to alteration of the tertiary structures of proteins towards an improved amphiphilic character,which is beneficial for PG to be used as an emulsifier or a foaming agent.Besides,PG has been demonstrated to be nonpathogenic,non-mutagenic and highly safe.Therefore,PG has received intensive attention especially in food industries as a food process aid.PG is produced by Chryseobacterium proteolyticum.However,the PG production rate of the current strain is too low,which hindered its industrialization and subsequently its applications.Therefore,the major purpose of this research is to engineer high PG-producing strains by mutagenesis.The characteristics of the selected high PG-producing strain were studied under different fermentation conditions,and the influences of ammonia on PG production in the selected strains was also explored.The result provided in this research laid a foundation for further study on the metabolic pathway of PG.1.Establishment of an immunological assay for the detection of protein-glutaminaseIn order to establish an effective method to quantitate PG,the mature peptide gene mpg was amplified from the genome of C.proteolyticum,and ligated with p ET32a(+)vector to construct a recombinant plasmid p ET32a(+)-mpg,which was then transformed into BL21(DE3).By isopropyl-?-D-thiogalactoside(IPTG)induction at 25°C for 6 h,the secreted recombinant protein m PG-His6 reached the peak expression level.After purification by nickel column,the recombinant protein was used as an antigen to immunize New Zealand white rabbits to prepare polyclonal antibody.The titer of the antibody was 1:5000 tested by ELISA,suggesting that it may be used in subsequent experiments.This polyclonal antibody was used to detect PG in the fermentation supernatant of C.proteolyticum by Western blotting.The results showed that the antibody had a good specificity and could be used for semi-quantitative detection of PG produced from C.proteolyticum.2.Engineer high PG-producing strain by successive mutation with combined mutagenesis methodAccording to the lethality rate and positive mutation rate,the mutation conditions were optimized and a protocol combined with successive ultraviolet(UV)photorepair mutagenesis and sodium nitrite-ultraviolet(NIT-UV)mutagenesis was established.The optimized condition of the UV photorepair mutagenesis was as follows: the concentration of strain was adjusted to OD600=1,the first UV irradiation time was 10 s,the photorepair time was 25 s,and the second UV irradiation time was 15 s.The optimized condition of NIT-UV mutagenesis was as follows: the concentration of NIT was 0.02 mol/L,the NIT treatment was 4 min,followed by UV irradiation for 6 s.Using NTG-Q0314 as the starting strain,5 successive rounds of UV photorepair mutagenesis and 5 successive rounds of NIT-UV mutagenesis were performed.A high PGproducing strain NIU-T1040 was finally obtained by screening from 17783 mutants with 48-well plate primary screening,48-well plate secondary screening,test tube culture and flask culture.Compared with the starting strain,the enzyme activity of the high PG-producing strain NIU-T1040 was 3.32 U / m L,which was 172.13% higher than that of the original strain(1.22U/m L).3.Characterization of the high PG-producing strain NIU-T1040The high PG-producing strain NIU-T1040 were characterized by growth and metabolism level,the gene level,the transcription level and the protein expression level.The results showed that the highest enzyme activity of NIU-T1040 is 1.79 U/m L under high ammonium condition.In the low ammonium environment,the PG enzyme activity of the starting strain NTG-Q0314 and the high PG-producing strain NIU-T1040 has been increased significantly.Meanwhile the enzyme activity of NIU-T1040 reached 3.37 U/m L and its tolerance to ammonia was greatly improved.The full-length sequencing of PG gene showed no mutation in PG gene itself.The results of PG transcription showed that low ammonia environment could improve both the transcription levels of PG in the starting strain NTG-Q0314 and the high PG-producing strain NIU-T1040,but still the transcription level of PG increased 4.86 times in NIU-T1040 at 16 h.In high ammonium environment,however,the transcription level of PG in NIU-T1040 was13.39 times higher than that of NTG-Q0314 at 16 h.The results of PG protein analysis also showed that the expression of PG protein in NIU-T1040 was significantly increased.4.Effect of ammonia on metabolic regulation of protein-glutaminaseBased on the observation of the effect of ammonium concentration on PG production,that high ammonium ion concentration inhibited the synthesis of PG rather than the activity of PG.The whole genome sequencing of wild-type strain QSH1265 was performed,and the main pathways of ammonia metabolism in nitrogen metabolism pathway was analyzed by KEGG.Three key speed limiting enzymes,the glutamic acid dehydrogenase(GDH),the glutamine synthetase(GS),the glutamic acid synthase(GOGAT)and an ammonia transporter(AMT)responsible for the transmembrane transport of ammonia inside and outside the cell were found.The real-time PCR showed that low ammonia environment could increase the transcription levels of AMT,GDH,GS and GOGAT,which would improve the ammonia metabolism regulation in high PG-producing strain and further promote its synthesis of PG.In this study,a combined mutagenesis protocol was established by optimization of the condition in a multi-step UV-photorepair mutation procedure and a multi-step NIT-UV mutation procedure.A polycolonal antibody against PG was prepared to improve the quantitation method of PG.As a result of the mutagenesis breeding method,a high PGproducing stain,NIU-T1040,was selected.The characteristics of this high PG-producing strain were thoroughly investigated including its growth and metabolism level,the PG gene level,the PG transcription level and the PG protein level.Moreover,the effect of ammonia concentration on the secretion of PG was investigated.My research laid a foundation for the future directed evolution of the strain and the rational optimization of the culture medium to further improve PG production.