Research On The Biosynthesis Process Of ε-Poly-L-Lysine About An Environmental Friendly Preservative

ε-Poly-L-Lysine is composed of 25³5 residue of L-lysine connect betweenα-carboxyl and ε-amino groups by dehydration condensation reaction.It had high security,wide antimicrobial spectrum and can be used in a wide pH range.The molecular weight of ε-PL is about 3500⁴500 Da.ε-PL is mainly used as a food preservative and also used in the field of biological materials and drug carrier.The production of ε-PL was already formed an industrial scale in Japan,but the related research in our country started fairly late.In order to improve the fermentation level,it’s essential to breed high-producing strain and optimize fermentation process,which also have a great significance to realize industrial production of ε-PL in our country.In this paper,Streptomyces albulus FQ-23 was used as the starting strain.Diethyl sulfate（DES）mutagenesis combined with resistance screening was used to breed high-producing strain,and then optimized its fermentation medium.The optimal feeding composition for ε-PL synthesis was studied by shake-flask feeding experiment,and the activities of key enzymes were determined.The optimal pH control technology was explored in a 5L fermenter during the bacteria growth phase.At the same time,differences in ε-PL production were investigated by feeding with distinct organic nitrogen sources.The main results are described as follows:（1）A mutant strain FQF-3 was obtained by DES mutation and resistance screening of glycine（Gly）,L-lysine（L-Lys）and sulfaguanidine（SG）.Its yield reached 0.976 g/L,which increased by 8.93% compared with the starting strain.（2）By screening a series of organic nitrogen sources,corn steep powder and fish meal were chosen as the mixed organic nitrogen source for ε-PL fermentation.The optimum concentration of carbon and nitrogen source was obtained by single factor experiment.Fermentation medium was optimized by Central Composite design（CCD）and response surface method.The optimization model was Y=1.370-0.006A-0.004B-0.010C+0.027D-0.006AB+0.005AC-0.004AD+0.010BC+0.009BD-0.007CD-0.083A2-0.087B2-0.081C2-0.110D2.The optimal fermentation medium（g/L）was: glucose49.70,（NH4）2SO4 9.95,corn steep powder 7.79,fish meal 12.52,KH₂PO₄·12H₂O 1.36,MgSO₄·7H₂O 0.5,K₂HPO₄·3H₂O 0.8,FeSO₄·7H₂O 0.03,ZnSO₄·7H₂O 0.04.The maximum yield of ε-PL reached 1.373 g/L,which increased by 40.7% than before optimization.（3）The effect of different feeding composition on ε-PL synthesis was studied by shake-flask feeding experiment.The result of feeding with glucose,（NH₄）₂SO₄ andyeast powder was the best,and the maximum production of ε-PL reached 1.75 g/L.On this basis,by changing organic nitrogen source into fish meal,the maximum yield of ε-PL reached 1.89 g/L.Corn steep liquor was compounded with fish meal in order to raise the concentration of bacteria.The result showed that when the concentration ratio of corn steep liquor and fish meal was 1:2,the maximum output of ε-PL was2.13 g/L,which increased by about 13% on the basis of feeding with fish meal.The activity of aspartokinase（ASK）and ε-PL synthetase（Pls）was determined after feeding with organic nitrogen source.The activity of ASK by feeding with mixed organic nitrogen source was correspond to feeding with fish meal,and it was higher than feeding with yeast powder.The activity of Pls was the highest by feeding with mixed organic nitrogen source,and it was 2.7 times than feeding with yeast powder and 1.2 times than fish meal.It was proved that the optimum way was feeding with mixed organic nitrogen source.（4）In the stage of growth of bacteria,12.5% ammonia was used to maintain the pH for 12 h when the initial pH of 6.8 naturally dropped to 6.5,6.0,5.5,5.0,respectively.It was determined that maintaining the pH of 5.0 was the most conducive to the growth of bacteria and accumulation of ε-PL.On this basis,ammonia was used to respectively maintain the pH for 6h,12 h,18h,when the initial pH of 6.8 decreased to 5.0.The result confirmed that it was the best way to maintain the pH of 5.0 for 12 h.The maximum production of ε-PL and concentration of bacteria reached 2.81 g/L and16.96 g/L respectively.（5）Based on pH control strategy,4 kinds of organic nitrogen sources,including yeast powder,fish meal,corn steep liquor and beef paste,were respectively batch fed at the same time batch fed with glucose and（NH₄）₂SO₄.The yield was the highest when feeding with fish meal.Its production was 29.30 g/L and increased by 10%compared with yeast powder.On this basis,the maximum yield reached 34.19 g/L by feeding with mixed organic nitrogen source of corn steep liquor and fish meal（the concentration ratio of corn steep liquor and fish meal was 1:2）.It was raised by 17%on the basis of feeding with fish meal.（6）In the stage of cell growth,feeding continuously with all ingredients based on pH control strategy,the final dry cell weight reached 53.78 g/L,the maximum production was 37.58 g/L and the yield was 4.42 g/（L·d）.