Construction Of The Engineering Bacteria With High Yield Of Alkaline Protease 2709 And Its Application Performance

Alkaline protease can maintain efficient enzyme activity under alkaline conditions and have a wide range of applications in detergents,leather,textiles and other fields.Among them,alkaline protease 2709 is one of the most widely used class of proteases in industry,and has a strong ability to decompose proteins.It is mainly produced through deep fermentation,extraction and refining by Bacillus licheniformis.However,its enzyme production was very low which cannot meet the needs of modern industrial development at present.This study was aimed to improve the yield of alkaline protease 2709 from Bacillus licheniformis through a multitude of methods,including ultra-violet mutation breeding,screening of a new promoter,construction of the engineering bacteria and so on.Furthermore,the enzymatic properties and washing performance of alkaline protease 2709 were also measured,and the research contents and the results are as follows:1.In the experiment of ultra-violet mutation breeding,the growth curve and lethality curve of the strain were detected which suggested that the best conditions was irradiated for 120 s.After incubated in light tight incubator,the ratio of the diameters of clear zones and colony diameter on milk agar plates was measured,and the bacteria whose ratio was the largest was separated.The yield of alkaline protease 2709 by this strain was 9186.6 U/mL contrasted with 5036.3 U/mL of the initial strain,in addition,it also had a good hereditary stability.2.As is known to us all,a promoter is the most important regulatory element for gene expression in microbial genetic system.A new type of promoter pChi was screened from the Bacillus licheniformis by SDS-PAGE and mass spectrometric detection.With the other two strong constitutive promoters pShuttle-09 and pyxiE as the control,pChi's expression activities in the alkaline proteases from B.clausii and B.licheniformis was investigated in B.subtilis WB600.The results showed that the functional expression of pChi was 1.25 times of pShuttle-09 and 2 times of pyxiE.3.The alkaline protease 2709 homologous expression vector,which was constructed using the shuttle vector pWH1520 and promoter pChi as expression regulatory elements,was transferred into the UV mutant through electrotransformation.Fermentation performance displayed that the yield of alkaline protease 2709 by this recombinant genetic engineering bacteria was up to 12888.6 U/mL,which was 155.9%higher than the original strain.4.Alkaline protease 2709 was produced by this engineering bacteria,and its enzymatic properties were studied,which showed that its optimum reaction temperature was 55?,and it had a good thermostability at low temperatures.Optimum reaction pH of alkaline protease 2709 was 10.0 in Gly-NaOH buffer,and it can hold efficient enzyme activity under alkaline conditions.Under the optimum reaction conditions,michaelis constant Km towards casein was 7.14 mg/mL and the maximum catalytic reaction rate Vmax was 10.63 mg/(mL·min).Furthermore,the study in its application performance suggested that alkaline protease 2709 had a good washing performance and stability as detergent additive.