Preparation Of Peptides From Crude Flax Proteins And Interaction Of Flax Trypsin Inhibitor(LUTI)with ?-Glucosidase

The partial hydrolysis of plant proteins by proteases can obtain biologically active peptides such as anti-oxidation,hypoglycemic,anti-tumor,anti-hypertensive,and activated cellular immune functions.It is the most popular research direction in the field of modern food and medicine,and also the main way to enhance the utilization value of plant proteins.Peptides from various plants are potential antioxidants,such as cottonseed protein,whey protein,soy protein and other hydrolyzed polypeptides,and antioxidant peptides exert their ability by scavenging free radicals.At present,the research on flax in China mainly focuses on the development and utilization of flaxseed oil,while the research on flax protein is less concerned.We used linseed meal as raw material to prepare antioxidant peptides,and compared the yield of different acid proteases and neutral proteases and the antioxidant capacity of peptides.The results were showed that using the 3.350 black yeast acid protease had the best enzymolysis efficiency with 28.52% polypeptide yield.Though the single factor and orthogonal experiments,the optimum conditions for the preparation of flax polypeptide were determined as follows: enzymatic hydrolysis temperature 60 ?,p H2.7,plus enzyme quantity is 6500u/g,enzymolysis time 2.5h,38.80% polypeptide yield.The molecular weight of the polypeptide was mainly distributed in the range of 1000 Da to 7600 Da by gel chromatography analysis.The enzymolysis flax polypeptide has good reducing power,with 89.99% DPPH radical scavenging rate for 1.5 mg/ml,and 85.57% superoxide anion clearance rate for 0.6mg/ml.Diabetes is one of the recognized health hazards.Controlling the activity of ?-glucosidase in the human gut is one of the effective methods for treating diabetes symptoms.Previous studies in our laboratory revealed that the flax trypsin inhibitor(Linum usitatissimum L.LUTI)is a polypeptide in linen that has dual activities of trypsin inhibitor and alpha-glucosidase inhibitor.To further investigate the molecular structure of the flax bifunctional polypeptide and the structure-activity relationship of glycosidase and trypsin activity,we chemically synthesized the flax bifunctional polypeptide gene and cloned into p GEX6p-1 vector for recombinant expression in E.coli BL21 to obtain soluble protein GST-LINUS-TI,it was cleaved by GST-Prescission Protease and purified by GST-Spharose4 B affinity column and Sephacryl S-200 gel column to obtain electrophoresis-purified r LUTI,and analyzed the inhibitory activity of r LUTI on double enzyme.The inhibitory activity of r LUTI on both enzymes was found to be significantly higher than the native LUTI.The relationship between flax trypsin inhibitor(LINUS-TI)and ?-glucosidase was analyzed by ?-glycosidase enzyme activity assay and gel filtration chromatography and dynamic light scattering(DLS).The results show that LINUS-TI not only It can inhibit the activity of inhibiting ?-glucosidase,and a complex can also be formed between LINUX-TI and ?-glucosidase.T44/A and K45/A mutants of r LUTI were obtained by site-directed mutagenesis.The inhibitory activity of the mutants to alpha-glucosidase was significantly decreased,and the inhibitory activity to trypsin was higher than that of natural LUTI and lower than that of r LUTI.Using the ZDOCK server for molecular docking between LINUS-TI and ?-glucosidase,12 amino acid residues of ?-glucosidase were found(Phe384,Asp392,Lys439,Asp568,Glu707,Asn708,Glu771,Asn772,Ser774,Gly780,Thr781,Glu784)interacts with the six amino acid residues of LINUS-TI(Ser1,Arg2,Arg3,Asp46,Phe47,Arg48)through 14 hydrogen bonds(H bonds).In order to verify the docking results,the LUTI gene and its point mutation or deletion mutant gene were connected to the p ET-3a vector to obtain the recombinant vector p ET-3a-LUTI and transformed into Escherichia coli strain E.coli BL21(DE3),and purified by Q-Sepharose ion exchange column and Superdex 75 gel column to obtain protein samples of electrophoretic pure wild type LUTI(r LUTI)and its mutants D46/A,F47/A,R48/A and deletion bodies r LUTI(2-69)? r LUTI(3-69)? r LUTI(4-69)?r LUTI(5-69).The inhibitory activity of r LUTI deletion bodies r LUTI(2-69),r LUTI(3-69),r LUTI(4-69)and r LUTI(5-69)on alpha-glucosidase was significantly decreased,and the inhibitory activity of trypsin slightly increased.The inhibitory activity of r LUTI mutants D46/A,F47/A and R48/A on alpha-glucosidase was significantly decreased,but the inhibitory activity of trypsin was lost.The above conclusions indicate that the N-terminal SXXXP [1-5] amino acid sequence of flax trypsin inhibitor plays an important role in maintaining its dual enzyme inhibitory activity,especially in the ?-glucosidase inhibitory activity.The 44,45,46,47 and 48 amino acids of r LUTI play different roles in inhibiting the activity of alpha-glucosidase and trypsinase.Among them,44 and 45 amino acids are the structural sites affecting the activity of alpha-glucosidase,while 46,47 and 48 amino acids are the structural sites affecting the activity of trypsinase.