Mutation And Screening Of Strain For Enhancing Curdlan Yield

Curdlan is an extracellular polysaccharide which is produced by Agrobacterium sp..Curdlan has good properties such as unique thermal gelling properties,water-holding capacities,the stability of freeze-thawing and safety.It will be widely applied in the food and biomedicine industry.At present,curdlan has been produced abroad in industrial scale,however the cost of production was high and the yield and quality of curdlan were needed to be improved.The modification of strain was critical step for improving the yield and quality of microbial product.In this study,yield and quality of curdlan was improved by inducing mutation of strain.The main contents included strain mutation and screening by UV mutation,NTG mutation,and combined mutation;metabolic characteristics of high-yield strain,and the structure and gel characteristics of curdlan from the high-yield strain.（1）By 16S r RNA gene sequence homology analysis and physiological and biochemical characteristics analysis,the original strain 714 was identified as Agrobacterium sp..（2）Agrobacterium sp.714 was mutagenized by UV.The seeds after 20h culture were diluted to OD600（10⁸CUF/ml）for mutation.The UV treatment time was 15s and 25s and the lethal rate was 74.1%and 97.7%.A screening system was established by combining the plate with aniline blue and shaking-flask fermentation.35 mutant strains was selected from plate with aniline blue and 10 positive mutant strains was further selected by shaking-flask fermentation.The high-yield strain 25-19-2 was selected and its crude yield of curdlan was 18.8g/L.The yield increased to 4.9times comparing with the crude yield of curdlan（3.8g/L）for parent strain.The heritability of strain 25-19-2 is stable.（3）When screening strains by the plate with aniline blue,the false positive results often appeared.The reason was that false positive strains produced lots acids resulting in the deep blue,but not curdlan.（4）The strain 25-19-2 was used for NTG mutagenesis.The 20h seed was mutated by NTG for 30min.The NTG dose was 0.4 mg/ml and 0.6 mg/ml and lethal rate was around 90%and100%.When lethal rate was above 90%,it was possible to get high yield strains.344 positive mutant strains were selected from plates with aniline blue,and 19 positive mutant strains with yield increased by 10%were selected by shaking-flask fermentation.The high-yield strain 0.6-45and 0.5-50 were selected and their crude and refined yield were 26.4g/L,19.4g/L and 26.5g/L,18.0g/L.The crude yield was 6.95 and 6.97 times of the crude yield of strain 714（3.8g/L）.（5）UV-Li Cl and UV-5-FU were used to treat the strain 0.6-45.4 positive mutant strains form UV-Li Cl and 17 positive mutant strains from UV-5-FU were selected from plate with aniline blue,however the high-yield strain was not be gotten after shaking culture.It was possible that the number of the selected strains were not enough.（6）The effect of glucose content in medium on the curdlan production and the sugar conversion by strain 0.6-45 and 0.5-50 were evaluated.When the glucose content in medium was below 90g/L,the both strains utilized the sugars almost completely and the residual glucose was1.11g/L and 1.05g/L.The crude yield of curdlan for strain 0.6-45 and 0.5-50 were 29.8g/L and30.0g/L after 4d fermentation.Metabolic characteristics of strain 0.6-45 and 0.5-50 were similar.At the 1^st day,the biomass reached highest,respectively 2.96g/L and 2.98g/L.The strain started to produce curdlan at 1^st day,the yield reached highest at the 4^th day.The crude yield was respectively 29.2g/L and 28.9g/L,and the refined yield was respectively 24.4g/L and 24.5g/L.The p H dropped lowest at the 1^st day,respectively 5.34 and 5.59,and then increased to 5.8 and 5.9 at the 3^rd day.The glucose content in culture medium dropped quickly to 3.14g/L and 4.50g/L respectively at the 3^rd day and then kept stable.（7）The structure and gel characteristics of curdlan from the high-yield strain were analyzed.The content of glucose in sample form strain 0.6-45 and strain 0.5-50 was 91.5%and 96.2%,and it showed the samples were pure.The FTIR and ¹HNMR showed the curdlan form strain 0.6-45and strain 0.5-50 wasβ-1,3 glucan.GPC results showed the molecular weights of curdlan（M_n）were respectively 1.44×10⁶ and 1.01×10⁶Da.The gum characteristics analysis shows that the gel strength of curdlan from strain 0.6-45 and 0.5-50 was lower than Japan sample,the fracture strength and fracture distance were higher than Japan sample.The TPA analysis showed that the hardness,gumminess and chewiness were lower than Japan sample,and the springiness,cohesiveness and resilience were higher than Japan sample.