| At present,bioactive peptides have attracted wide attention due to their nutritional,functional and safety characteristics.As one of peptide preparation methods,microbial fermentation method uses the protease produced by microorganisms in the fermentation process to degrade the substrate protein,and the enzyme production and enzymatic hydrolysis steps are carried out simultaneously,which simplifies the process and has large treatment capacity.However,the disadvantage of this method lies in the fact that in the growth process of the strain used,it not only produces protease,but also consumes a lot of peptides as nutrients,which leads to the low yield of final peptides and seriously affects its popularization and application.Mutation breeding is the main method to obtain high-yield,low-consumption and high-quality strains.In recent years,adaptive laboratory evolution(ALE)as a mutation breeding method has been concerned.In order to improve the breeding efficiency,a new method of ultrasound-assisted adaptive evolutionary mutation(US-ALE)was proposed.First of all,through the introduction of classical reversion mutation test(Ames test),the parameters of ultrasound promoting its reversion mutation were optimized,the types and rules of gene mutation were studied,the mechanism of ultrasonic mutation was revealed,and the ultrasonic evolution mutation system was constructed;the ultrasonic evolution mutation of Bacillus was carried out,and the mutant strains with high-yield peptide were screened,and the fermentation effect was evaluated.The main contents and results were as follows:(1)Ames strain was used as model organism to study the types and rules of ultrasound-mediated gene mutation,explore its mutation mechanism,and construct ultrasound-mediated evolution mutation system.1)Optimization of parameters of ultrasound promoting the reversion mutation of Ames strains: Ames strains TA97 a,TA98,TA100 and TA102 were applied as model organisms and the reversion mutation rate was used as an index to explore the influences of different ultrasound parameters on the reversion mutation.The results displayed that the reverse mutation rates of TA98 and TA100 were significantly increased after sonication,while the reverse mutation rates of TA97 a and TA102 were not significantly changed.The absolute reverse mutation rate of TA98 increased by 155.58%(frameshift mutation)over the control when it was sonicated at power density of 20 W/m L and frequency of 40 k Hz for 5 min after cultivated for 6 h.The absolute reverse mutation rate of TA100 increased by 281.20%(base substitution)over the control when it was sonicated at power density of 40 W/m L and requency of 35 k Hz for 10 min after cultivated for 6 h.2)Sequencing analysis of frame shift mutation of ultrasound-mediated TA98revertants: Based on the sequencing results of 1752 TA98 revertants,127 types of reversion mutation were found(all occurred in the core mutation region: from the 832 th bp to 915 bp of original gene,referred to as No.832-915),among which the types of reversion mutation unique to sonication group were 36 more than the control.The +1 bp insertion,as a classical reverse mutation,accounted for 17.81% and 12.45% of all frameshift mutations in the control and sonication group,respectively.However,the proportion of TA98 strain reverse mutation to the wild-type sequence(CC?CCC)was in minority,in which the sonication and the control group accounted for 0.34% and 0.23%respectively.In addition,the-2 bp deletion was the main type of frameshift mutation in both the control and sonication group.In the non-classical reverse mutation,the additional-29 bp deletion(1.60%)and +7?+43 bp insertions of direct repeat sequences(16.10%)were observed in TA98 revertants,of which the percentages of +16 bp(4.22%)and +28 bp(2.85%)were significantly higher than those of adjacent other base insertions.These results indicate that ultrasonic treatment not only increased the probability of frameshift mutation,accompanied with many unique types of reversion mutation,but also promoted the insertions and deletions of DNA large fragments.3)Sequencing analysis of base substitution of ultrasound-mediated TA100revertants: Based on the sequencing results of 196 TA100 revertants,five mutation types(CCC?CTC/CAC/TCC/ACC/GCC)were found,among which CCC?CTC(classic mutation type)accounted for the highest proportion(35.00%)in all base substitutions.After sonication,the proportion of non-classical mutation type CCC?CAC was the most(37.33%),while the classic mutation type CCC?CTC accounted for 17.00%.Besides,the unique non-classical mutation type CCC?GCC was found in the sonication group,which accounted for 14.33%.These results demonstrated that sonication not only increased the probability of base substitution and decreased the proportion of classical mutation types,but also promoted the occurrence of base transversion(C?G)in base substitutions.4)Studies on mechanisms of ultrasonic mutation and the establishment of evolution mutation system: Based on the analysis of transcriptomics of ultrasound-mediated frameshift mutation of TA98 revertants,45 differentially expressed genes(29 up-regulated and 16 down-regulated)were observed after sonication,which did not cause the changes of gene expression by a large margin.This suggested that the ultrasound mutation belonged to weak treatment,which was suitable to be applied to adaptive evolution as an external mutagenic physical field.Combining with transcriptome analysis and Lee's hypothesis,it was speculated that ultrasound may cause the DNA replication to be blocked instantly.Under the effect of ultrasound-mediated "heat breathing",single-stranded DNA forms hairpin structure by itself and carries out micro-homologous recombination with complementary strand,and then DNA initiates related repair mechanism to enable it to continue DNA replication.Based on the hypothesis of "heat breathing" induced by ultrasound,the cold-shock treatment was introduced to optimize the mutagenic system.The best effect was enhanced by ultrasound-mediated frameshift mutation(reversion mutation rate increased by 120.83%)when cold-shock time was 1.5 min.Besides,the best effect was enhanced by ultrasound-mediated base substitution(reversion mutation rate increased by 43.82%)when cold-shock time was 2.5 min.In the simulation test of single-gene evolutionary mutation,the significant enrichment effect was observed in positive mutants and its proportion was more than 95% after two times of transferring strains.(2)The fermentation strains with high-yield enzyme and low-consumption peptide were selected from 8 kinds of Bacillus as the original strains;based on the ultrasonic-assisted adaptive evolutionary mutation(US-ALE)system,the mutagenic strains that can adapt to the medium with inorganic nitrogen and carbon source were screened;the nitrogen transformation and the activity changes of related enzymes during fermentation were studied through the evaluation of fermentation performance,so as to effectively improve the yield of peptides.1)Screening of high-yield protease and amylase producing strains: The protease activity,amylase activity,biomass,peptide conversion rate and bacterioprotein conversion rate of 8 Bacillus strains on casein medium and starch medium were compared.The results showed that the biomass of Bacillus No.1(Bacillus velezensis),Bacillus No.4(Bacillus amyloliquefaciens)and Bacillus No.8(Bacillus amyloliquefaciens)on casein and starch medium was higher than other strains,accompanied with the higher activities of amylase and protease on the two media.In general,No.1(Bacillus velezensis)was selected as the starting strain for subsequent evolutionary mutation.2)Ultrasound-assisted adaptive evolution mutation of starting strain: Based on the parameters of ultrasound evolution mutation system,the starting strain was mutated for 40 days.With the decreased proportion of casein medium(from 50% to 0.5%)and the increased proportion of starch medium(from 50% to 99.5%),the p H value of fermentation broth decreased faster in US-ALE group compared with ALE group.Based on the comprehensive indices of biomass,protease activity,amylase activity and peptide content,two mutant strains of US-ALE-BV3(mutant strain 1)and US-ALE-BV22(mutant strain 2)with excellent characteristics were selected from 30 mutants,and used for the evaluation of subsequent fermentation performance.3)Comparisons of nitrogen transformation and sugar metabolism between original strain and mutant strain: According to the change of growth curve and p H value of fermentation broth,the growth trend and fermentation performance of mutant strains changed significantly.Further combined with the nitrogen transformation,sugar source metabolism and the related enzyme activity were,the ability of mutant strain 1 to utilize inorganic nitrogen,organic nitrogen and sugar sources was significantly enhanced compared with the original strain.The ability of mutant strain 1 to synthesize protein-N was significantly higher than that of mutant strain 2.Compared to the original strain,the<3K peptide-N content of mutant strain 1 and mutant strain 2 increased by 29.81% and66.07%,respectively,after 24 h fermentation.Besides,the total peptide-N content of mutant strain 2 increased faster than mutant strain 1.In conclusions,mutant strain 1 has a strong ability to synthesize bacterioprotein using inorganic nitrogen,while mutant 2prefers to use inorganic nitrogen to synthesize peptides.4)The further improvement of peptide yields by cell autolysis: Bacterioprotein was considered as a good protein source.Under the optimal autolysis conditions(autolysis temperature of 50?),the content of autolytic peptide-N of the fermentation broth based on the mutant strain 1 and mutant strain 2 increased by 30.40% and 19.67%,respectively compared to the original strain.Moreover,the amino acid composition of the fermentation product was improved due to the introduction of bacterioprotein.In the actual fermentation experiments of soybean meal and corn gluten meal,the peptide yield of mutant strain 1 respectively was 4.07 times and 7.09 times higher than that of the original strain,and the peptide yield of mutant strain 2 was 3.35 times and 5.49 times higher than that of the original strain,respectively.Therefore,in this study,mutant strain1 was better than mutant strain 2. |
PDF Full Text Request