Study On Hypoglycemic Polypeptides In Carrots

Diabetes mellitus(DM)is a disease of glucose,protein and fat metabolism disorder caused by insulin utilization disorder or insufficient secretion,which has become one of the hidden killers threatening human health.In a number of hypoglycemic drugs,?-glycosidase inhibitor drugs as a kind of oral fall blood sugar,can inhibit the hydrolysis of?-glucosidase on disaccharides or oligosaccharides 1?4 glycosidic bonds in the brush border epithelial cells of human intestinal mucosa,thereby effectively reducing blood sugar levels after meals,has a good effect on type 2 diabetes.In recent years,the novel?-glucosidase inhibitors screened from natural products at home and abroad mainly include polysaccharides,alkaloids and glycosides.Although the preparation,screening and separation of hypoglycemic peptides have been reported,the molecular targets are not clear.In the early stage of our laboratory,we used?-glucosidase as a target to screen hypoglycemic polypeptides,it was found that the trypsin inhibitor LUTI of flax potato I also has?-glucosidase inhibitory activity.Carrots,as one of the most widely planted vegetables in the world,are rich in vitamins,carotene,volatile oil,coumarin and other nutrients.At present,the reasearch have shown that some biologically active substances in carrots,namely phenolic compounds(especially Chlorogenic acid),carotenoids,polyacetylene and ascorbic acid(vitamin C)have anti-cancer,antioxidant,anti-inflammatory,antibacterial,plasma lipid modification and blood lipid formation effects,which have the potential to improve human health.However,there are few studies on carrot protein and peptides.Therefore,on the basis of LUTI's work,we took carrots as the research object to explore whether there are polypeptides in carrots that inhibit?-glucosidase activity.The research content mainly includes the following parts:1.Purification and recombinant expression of pathogenesis-related proteins in carrot.The carrot juice obtained by dicing,squeezing,centrifuging and filtering,then separated and purified by an anion exchange column and a gel column to obtain a polypeptide about 16k Da.Through mass spectrometry analysis,it is known that the polypeptide is a carrot pathogenesis-related proteins called Dau c1.Then the Dau c1protein was recombinantly expressed in E.coli and purified.Furthermore,we analyzed the inhibitory activity of natural and recombinant protein on?-glucosidase,It turned out they all do.2.The expression,purification and characterization of the trypsin-like inhibitor DCHP in carrot.Putting LUTI into the NCBI database for homologous sequence comparison,we found that an unnamed hypothetical protein of carrot has 85%similarity with LUTI.We named it DCHP(Daucus Carrot hypoglycemic peptide).Afterwards,the DCHP gene was recombinantly expressed in E.coli,and purified by Ni²⁺-NTA affinity column and Superdex 75 prep grade gel column to obtain electrophoresis pure DCHP with a molecular weight of 8k Da.The pure DCHP was crystallized and analyzed,we obtained the structure information of DCHP.Analysis of the inhibitory activity of DCHP's?-glucosidase,trypsin and chymotrypsin shows that DCHP has?-glucosidase inhibitory activity,but does not have the inhibitory activity of trypsin and chymotrypsin.DCHP maintained good stability at 50°C for120 minutes,and its hypoglycemic activity did not decay,and its optimal p H for inhibiting glycosidase is 8.0.Incubate DCHP with pepsin,trypsin,and chymotrypsin for 120 minutes at room temperature.DCHP was incubated with pepsin,trypsin,and chymotrypsin at room temperature for 120 minutes,and then protein electrophoresis showed that DCHP can tolerate trypsin and chymotrypsin,but not pepsin.Finally,the content of glucose and lactose in Caco-2 and Hep G2 cells after DCHP incubation with different concentration gradients was detected,revealed that DCHP has effects of lowering blood sugar on the cell model.3.Molecular docking and biochemical verification of DCHP and?-glucosidase.By homologous sequence alignment between DCHP and LUTI and molecular docking analysis between DCHP and?-glucosidase,we made 8 mutations to DCHP,which were Q49/A,Q49/E,Q49/K,Q49/L,Q49/N,Q49/Y,E43/A,T48/A.The results showed that the inhibition rate of?-glucosidase in DCHP E43/A and DCHP T48/A mutants was significantly decreased,indicating that amino acids at 43th and 48th are the key sites for inhibition of?-glucosidase activity,and Glu mutated to Lys at the 49th position,DCHP has a strong trypsin inhibitory activity.These results founded that two functional polypeptides with?-glucosidase inhibitory activity in carrots,which are expected to be used as new?-glucosidase inhibitors to regulate blood glucose levels.It provided a new idea for developing carrot health functional food.