Studies On Microbes Producing 1-deoxynojimycin

1-deoxynojirimycin is an alpha-glycosidase inhibitor, which can inhibit the absorption of sugar, and then lower the blood glucose effectively. Mulberry has an excellent hypoglycemin effect as a traditional Chinese medicine, and its active composition has been identified as DNJ. Former studies has determined the DNJ content from these materials as follows: different mulberry varieties,different leaf positions, different silkworm species as well as silkworm body, silkworm excrement and pupa in different developing stages. At present, the silkworm and mulberry resources have been developed into hypoglycemic products in clinical practice.Now DNJ and its analogs have been detected from many microorganisms and plants. Mulberry has the highest DNJ content among the plants and the DNJ of the silkworm is obtained from mulberry. Although there is no conclu- sion of the exact origin of DNJ in mulberry, people generally consider that DNJ is synthesized by the mulberry. Meanwhile some studies show that many secondary metabolism products are generated by the associated microorganis- ms in the plants and DNJ is firstly discovered in Streptomyces. In order to explore the DNJ origin, we have isolated the endophytes in mulberry, and then determined the DNJ content of the endophytes by RP-HPLC-UV. We have discovered the endophytes that could produce DNJ. Further researches were as follows: the identification of the endophytes producing DNJ stably, the optimization of fermenting conditions, the initial identification and character- ization of DNJ produced by the endophytes. At last we studied the security and the hypoglycemic effect of the fermentation broth.1 The endophytes from mulberry, tissue cultured seedlings and mulberry seeds were isolated and determined the DNJ content with the method of RP-HPLC-UV. Nine endophytes producing DNJ were obtained, clustered into seven endophytes by the analysis of rep-PCR. Among of the seven endophytes, strain Y1 and Y2 showed stable quality in the DNJ production.2 Taxonomic determinations of bacteria strain Y1 and Y2 were carried out, including morphological observation, physiochemical analyses, test of biolog- ical antomated bacterial identification system, analysis of the fatty acids and 16S rDNA sequence analyses. The results showed strain Y1 was fell to Stenotrophomonas maltophilia and its 16S rDNA sequence was registered in GenBank (GenBank accession NO.GQ268318); Strain Y2 fell to Micrococcus luteus and its 16S rDNA sequence was registered in GenBank (GenBank accession NO.GU591156).3 Through the cultivation in shake-flask fermentation, the effects of environmental conditions and nutrition of strain Y1 producing DNJ were studied.The optimal shaking flask fermentation conditions were confirmed by one-factor-at-a-time experiment. The optimized fermentation conditions for DNJ production were: inoculum volume 2%(v/v), solution amount 50mL/250mL, fermentation temperature 30℃, initial pH6.0.Based on the results above, the effects of nutrition on production of DNJ were studied involving the optimal carbon, the optimal nitrogen, C/N, inorga- nic salt by one-factor-at-a-time experiment. In the optimization of medium, the influences of corn meal, maltose, peptone, urea, MgSO4, (NH4)2SO4 and NaCl on DNJ production were first evaluated using Plackett Burman design. Among of the test components, the effect of peptone and MgSO4 both reach extremely significant level and the urea shows significant effect on the DNJ production. However, maltose and NaCl have no significant effects on DNJ production. The path of steepest ascent was used to approach the optimal region of the medium composition. In the last step, the optimal concentrations of test components were determined by central composite design and response surface analysis.The optimum fermentation medium for production of DNJ were: maltose 40.0g/L, peptone 14.285 g/L, urea 7.391 g/L, MgSO4 0.7706 g/L, NaCl1g/L,corn meal 20.0g/L, maltose 20.0g/L, soybean meal 29.5g/L, (NH4)2SO4 1.0g/L, CaCO3 3.8g/L.Based on the optimal fermentation condition and medium, the spectrum peak area of DNJ after derivatization reached 102.9mAU*min, 227.21% higher than that of basal medium.4 The initial extraction, purification of DNJ produced by strain Y1 was studied by Rep-HPLC-UV and MS analysis. The results showed that the DNJ produced by strain Y1 was initial purified. The analysis of MS showed that the DNJ produced by Y1 had similar properties to the DNJ from Mulberry leaves. However, there were differences between the DNJ extraction and DNJ standard sample. It was concluded that the DNJ from strain Y1 and Mulberry leaves was analogue of DNJ sample.5 The security and hypoglycemic effect of fermention broth were studied by animal experiments. The results showed that the fermention broth was safty for test mice and had excellent hypoglycemin effect, so it could relieve diabetes symptoms effectively.