| Putrescine,also named as 1,4-diaminobutane,has great industrial value.Nylon-4,6 is the most noteworthy applications produced by polycondensation of putrescine and adipic acid.At present,the synthesis of putrescine for industrial use mainly depends on chemical method.Unfortunately,the raw materials of this route are non-renewable petroleum products which are unsustainable.At the same time,the conditions of chemical reaction are relatively stringent with expensive catalyst.Therefore,it is necessary to find a green and efficient way to produce putrescine.People turned their attention to the biosynthesis of putrescine,hoping to convert renewable raw materials such as glucose into putrescine by microbial fermentation which was sustainable and with less pollution.In recent years,microorganisms have been widely used by researchers to produce putrescine.But there are still many chanllenges.The culture broth of biosynthetic process contains various substances containing amino-group such as proteins,amino acids,polypeptides and amines,thus probably interfering the putrescine determination by HPLC.How to accurately evaluate the production performance of strains has become an urgent problem to be solved.In addition,the yield of putrescine produced by microorganisms depend on the natural synthetic pathwayu is low,which can not meet the needs of industrial production.To solve the above problems,the research was conducted from the following three aspects:?1?In previous experiments,it was found that when dansulyl chloride was used as derivatization reagent in the quantitative determination of putrescine,the complex components in culture broth would seriously interfere with the detection,resulting in significant deviation of the results.By analyzing the influence of the components in culture broth on the detection of putrescine,the calibration curve was established to eliminate the errors in the routine detection process.The detection error of the improved method was less than 7.96%,which improved the accuracy of detection.?2?By increasing the expression level of two key enzymes,arginine decarboxylase?speA?and agmatinase?speB?,for the synthesis of putrescine from arginine?ADC pathway?,the transformation efficiency of arginine and the accumulation of putrescine can be enhanced.In order to regulate the expression level of key genes in putrescine biosynthesis pathway,three expression plasmids with diffrent copy number?p COLADuet,pETDuet and pRSFDuet?were chosed to adjust the expression level of the target genes.The results of fermentation experiments showed that the low-copy plasmid?pCOLADuet-speA-speB?and medium-copy plasmid?pETDuet-speA-speB?had positive effects on the enhancement of putrescine production,high-copy plasmid?pRSFDuet-speA-speB?had no significant effect on the increase of putrescine.And the production performance of the strain carrying medium-copy plasmid was the best,the putrescine was increased from 1.64 g·L-11 to 3.94 g·L-1.Meanwhile,the expression level and catalytic efficiency of the key enzymes in PUT2 were higher than those of the low copy or high copy expression plasmid.?3?In order to further improve the yield of putrescine,the optimum fermentation medium and culture conditions were determined as follows:SOB medium with 8 g·L-11 glucose,glucose and arginine were added during the fermentation,and the agitation rate and the ventilation were set as400 r·min-1 and 1 vvm,respectively.The titer of putrescine in intermittent fed-batch fermentation were 26.21 g·L-1. |
PDF Full Text Request