| L-asparaginase(EC 3.5.1.1),belongs to a family of amidohydrolases that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia.L-asparaginase(EC 3.5.1.1)showed great commercial value owing to its effective treatment of acute lymphoblastic leukemia(ALL),Hodgkin disease,breast cancer and pancreatic cancer.L-asparagine is one of the nutrients for cancer and normal cells.Because these cancer cells lack the of L-asparagine synthetase activity,it can be starved by L-asparaginase hydrolyzing L-asparagine in the blood,which showed little effect on normal cells.In addition to its clinical application,the enzyme was also used as a promising acrylamide-mitigating agent to produce acrylamide-free food products by removing asparagine(one major precursor of acrylamide)without any changes in appearance,sensorial properties,flavor and nutrition.Currently,numerous L-asparaginases have been widely found from a variety of natural sources(microorganisms,animals and plants),in which the microbial L-asparaginases are the promising source for L-asparaginase production in large scale for application in clinical and food industry.However,the current commercial L-asparaginase was mainly derived from Escherichia coli and Erwinia,and the commercial L-asparaginase is mainly monopoly by foreign companies(Novozymes and DSM).It is urgent to explore the relevant research in China.Considering the food safety,the E.coli was not suitable for the production of L-asparaginase.Therefore,B.subtilis with GRAS(Generally regarded as safe)status and strong secretion ability of recombinant proteins was the most suitable host for the production of proteins used in the food and pharmaceutical industry.In this study,a type I L-asparaginase gene from Bacillus licheniformis Z-1(BlAase)was cloned and BlAase was expressed in B.subtilis RIK 1285.The secretion pathway was studied in details.Furthermore,the production level of BlAase was further enhanced through the modification of the expression elements.The main results of this thesis are summarized as follows:1.Secretory expression of BlAase in B.subtilisIn this study,the gene encoding BlAase was successfully cloned and BlAase was expressed in B.subtilis RIK 1285.Results showed that even without the mediation of any N-terminal signal peptides,BlAase can efficiently secrete into the medium.After72 h of fermentation,the intracellular and extracellular enzyme activities of BlAase reached 215.41 and 107.16 U/m L.Furthermore,significant intracellular enzyme activity was detected after 12 h of cultivation and reached a plateau at 348.3 U/m L after 24 h of cultivation,followed by a slight decrease in the subsequent 72 h of cultivation.By contrast,the extracellular BlAase activity was detected after 48 h of cultivation.After entering into mid-stationary growth phase,the extracellular enzyme activity sharply increased over time.After the cultivation,the final BlAase activity reached at 426 U/m L,with intracellular and extracellular enzyme activity reached147.02 and 279.03 U/m L,respectively.As the secretion of BlAase without the mediation of any N-terminal signal peptides.In order to make it clear whether the N-terminal sequence of the BlAase was involved in direction of secretion and removed during the translocation process,the N-terminal amino acid sequences of the BlAase were sequenced.Results showed that both the intracellular and the extracellular BlAase expressed in B.subtilis RIK 1285 have the same amino acids(MKKKVALITT),identical to the amino acids deduced from the nucleotide sequences,further confirming that BlAase itself is free of signal peptide.Altogether,the results preliminarily indicated that the BlAase was expressed and released to extracellular milieu via non-classical secretion pathway in B.subtilis.2.Study on the secretion pathway of BlAase in B.subtilisIn order to prove that BlAase is secreted via non-classical secretion pathways,cell lysis and classical secretion pathways(Sec and Tat secretion pathways)must be excluded.Studies showed that the secretion of BlAase was not simply caused by cell lysis.Further investigation indicated that the secretion of the BlAase was via neither Sec-nor Tat-dependent secretion pathway,and both the N-and C-terminal regions of the BlAase were essential for its expression and secretion,implying that BlAase might be secreted via a non-classical secretion pathway.To explore its secretion ability,BlAase was used as a signal peptide to direct the secretion of various heterologous proteins,where two of five proteins were successfully secreted with the mediation of BlAase.To the best of our knowledge,this is the first time to achieve extracellular expression of L-asparaginase via non-classical protein secretion pathway in B.subtilis,which could provide a potential tool for secretory expression of recombinant proteins in B.subtilis using BlAase as a signal peptide.3.Optimization of the expression level of BlAase in B.subtilis based on the original expressionThe BlAase has been successfully expressed and secreted in B.subtilis RIK 1285,However,the quite low yield hindered its industrial application.Thus,in this study,a combined strategy was performed to enhance the BlAase production in B.subtilis.(i)Fifteen single strong promoters were chosen to replace the original promoter P43,and the promoter Pyvy D showed the best performance among these 15 promoters,with the enzymatic activity reached 436.28 U/m L,223.96 and 212.32 U/m L for intracellular and extracellular enzyme activity,respectively,after 72 h of cultivation at 37 ℃,which was 1.25 times higher than that of control strain with promoter P43.(ii)The dual-promoter systems were constructed with four promoters(Pyvy D,P43,Papr E,and Pspo VG)to enhance the BlAase production,and the BlAase activity reached 502.11 U/m L with the promoter pair Papr E-Pyvy D.Furthermore,the BlAase activity was further enhanced to 568.59 U/m L by the modification of core regions of promoter Papr E-Pyvy D,which was 1.63 times higher than that of control strain with promoter P43.(iii)After replacing the ribosome binding site(RBS)sequence of promoter Pyvy D,the BlAase activity reached 790.1 U/m L,which was 2.27 times higher than that of the original strain containing promoter P43.(iv)The BlAase expression level arrived at 2163.09 U/m L in a 10 L fermenter after 36 h of cultivation,which was 6.2 times higher than that of the original strain contains with promoter P43.These results above indicated that the transcription and translation combined strategies were efficient to enhance the production of recombinant proteins. |
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