Study On Low-endotoxin Strain Producing Biomaterial Polyhydroxyalkanoates

Polyhydroxyalkanoate(PHA)is a biopolyester material produced by microorganisms using different carbon sources for aerobic fermentation,and its production cost is high.At the same time,most of the traditional synthetic carriers of PHA are commercial engineering bacteria,which can synthesize heat sources such as endotoxin,and the synthesized PHA needs to be purified several times to remove the heat source before it can be used in the field of human health such as medicine,which further increases the cost of PHA.In order to reduce the production and purification cost of PHA,we introduced the PHA synthetic plasmid pBHR68 into a low endotoxin strain(Clear Coli BL21),and synthesized PHA based on this platform.After testing,compared with the traditional PHA synthetic strain E.coli BL21(pBHR68),the endotoxin content of the Clear Coli BL21(pBHR68)strain was reduced by about 110-fold to 7.915pg/m L.The endotoxin content in this PHA is similar to the endotoxin concentration in human plasma under normal circumstances,and within the tolerance range of the human body,the toxic and side effects caused by direct use are small.This result indicates that the low endotoxin strain Clear Coli BL21 as the starting strain can greatly reduce the endotoxin content in the production of PHA,which is expected to simplify the subsequent endotoxin removal process and achieve the purpose of reducing production costs.In addition,this topic also explored the growth characteristics of Clear Coli BL21(pBHR68)strain,the production of PHA and the content of PHA after extraction and purification.The results showed that the growth trend of Clear Coli BL21(pBHR68)strain and E.coli BL21(pBHR68)strain was similar under the condition of sufficient nutrients.The PHA synthesized in bacterial cells was detected by confocal scanning electron microscopy and transmission electron microscopy,and a low-endotoxin-producing strain of PHA was successfully constructed.After extraction and purification,the content of PHA was about7.667wt%.In order to further improve the yield of PHA,we tried to study from two aspects:optimization of fermentation process parameters and selection of excellent strains by radiation mutagenesis.In terms of optimizing fermentation process parameters,we explored the effects of temperature,inoculum size,glucose concentration,fermentation volume(dissolved oxygen),and inducer concentration on the fermentation of Clear Coli BL21(pBHR68)strain.The data showed that 37°C,5% inoculum,20g/L glucose concentration,50 m L fermentation volume,and IPTG inducer concentration of 0mmol/L were more conducive to the accumulation of bacterial cells and the synthesis of PHA during the fermentation process.In terms of selecting excellent strains by radiation mutagenesis,according to earlier reports,it can be seen that the strain will undergo morphological changes in a high-salt environment,which is beneficial to increase the content of PHA.The Clear Coli BL21(pBHR68)strain was subjected to different doses of radiation mutagenesis,followed by determination of the survival rate of the strains and screening of PHA-producing strains in extreme environments.The Clear Coli BL21(pBHR68)salt-tolerant strain(Clear Coli BL21(pBHR68)-H)was preferentially selected,and by detecting its growth curve,endotoxin content and PHA production,it was found that the growth of the strain after irradiation was poor,but the endotoxin content did not change significantly compared with the strain before irradiation.And by scanning electron microscope and laser confocal scanning electron microscope,the morphology of the strain changed from short rod to long strip,which is expected to increase the accumulation of PHA.In conclusion,this study successfully constructed a PHA low-endotoxin-producing strain Clear Coli BL21(pBHR68),which effectively reduced the cost of endotoxin removal,but after fermentation,extraction and purification,the obtained PHA yield was low.By optimizing the fermentation parameters and radiation mutagenesis to select excellent strains,the optimal fermentation process parameters were finally determined,and the long-shaped Clear Coli BL21(pBHR68)-H strain was screened,which is expected to further improve the production of PHA.