Construction And Fermentation Process Control Of The Lumbrokinase Engineering Bacteria

Lumbrokinase(LK)is a group of serine proteases found in earthworms that have both fibrinolytic activity and kinase activity.The enzyme can stimulate fibrinolytic enzyme to convert into fibrinolytic enzyme,and thus catalyze fibrinolysis,which can contribute to thrombolysis.It has been widely used with the characteristic of good stability and its enzyme preparation can be taken both orally and injected.At present,the lumbrokinase gene has been expressed successfully in fungal,bacteria and animal cells,but the expression is not satisfactory.In this paper,wild earthworms were selected from the soil,and the species was identified as Eisenia Foetida,then the gene lks2 was cloned from it.The recombinant bacteria GG799-p KLAC1-lks2 and GS115-p PIC9K-lks2 were successfully constructed with the Kluyveromyces lactis and Pichia pastoris as the host.Through high density fermentation and process control,the high expression of lumbrokinase in Pichia pastoris was successfully achieved,and the maximum enzyme activity was 1140.8 U/m L.The main work is as follows:(1)Wild earthworms were screened from the soil of the campus,and the molecular biology was used to identify it as Eisenia Foetida.The total RNA of it was extracted and c DNA was obtained by reverse transcription,and then the gene lks2 was cloned.Genetic analysis was carried out with DNAMAN and other bioinformatics software.The total length of the gene was 738 bp,and encoding 245 amino acids.It was 99.59% homologous with the reported F238(Gene Bank No.DQ202401),with the difference between the 46 and 59 amino acids encoded in the 136,175 and 398 bases.The structure modeling and substrate docking were performed on the lumbrokinase LKS2,and the catalytic activity center of it was predicted(Tyr167-Ile218-Gly219-Asp189-Gly219).(2)The recombinant plasmid p KLAC1-lks2 was successfully constructed and expressed in the Kluyveromyces lactis GG799.After optimization,the optimal culture medium for recombinant bacteria was: glucose 25.0 g/L,yeast extract 10.0 g/L,tryptone 20.0 g/L and KH2PO4 10.0 g/L.The enzyme activity was the best under this condition,and the enzyme activity peaked to 140.4 U/m L at 72 h.(3)The recombinant plasmid p PIC9K-lks2 was successfully constructed and expressed in the Pichia pastoris GS115.By optimizing,the best induction conditions were: initial bacteria volume OD600 = 1.5,p H 5.5,and the temperature 28?.In addition,the addition of 0.1% serine can effectively improve the enzyme production capacity of recombinant bacteria.Under the optimal conditions,the lumbrokinase activity could reached a peak of 397.6 U/m L after methanol induction for 84 h,and increased by 56.3%.(4)On the basis of the shake bottle optimization,the high density fermentation conditions of the recombinant bacteria GS115-p PIC9K-lks2 were optimized using 10 L fermentation tank,and the optimum inoculation concentration was 9±0.5 g/L.In addition,the addition of 0.5% yeast extract can promote the production of enzyme.With the control of fermentation process,the activity of lumbrokinase was up to 1140.8 U/m L,which was 11.5 h shorter than the pre-optimized period,and enzyme activity increased by 1.16 times.