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Study on the Nutritional Evaluation and Trypsin Inhibitor of Wild Soybean (Glycine Soja Sieb.Et Zucc

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Tutor: ZhangLi
School: Ocean University of China
Course: Aquatic Products Processing and Storage Engineering
Keywords: Wild soybean,nutritional value,trypsin inhibitor,purification,geneticcloning
CLC: S565.1
Type: Master's thesis
Year:  2013
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Abstract:
China is a country with the largest distribution of wild soybean in the world. Wild soybean has rich variation with potential application value, which is lack in cultivated soybean. Wild soybean is also an important genetic source of the crop improvement. The protein content of wild soybean seed is higher than that of the cultivated soybean.Wild soybean has strong resistance and adaptability, which may be related to the higher expression quantity of trypsin inhibitor(TI) of wild soybean than that of the cultivated soybean. In view of the high expression and good biological activities of wild soybean trypsin inhibitor(WSTI), it is necessary to carry out systematic study. This study will deepen the understanding of Jimo wild soybean resources, which will provide a certain theoretical basis and data for the reasonable protection and utilization of the wild soybean resources.In this study, the nutritional value of Jimo wild soybean was discussed firstly. Then, the active ingredient of WSTI was separated and purified from wild soybean by gel filtration chromatography and affinity chromatography, and then the structural properties for WSTI were considered to some extent. Meanwhile, the activity of the trypsin inhibitor was determined by the established fluorescence spectrophotometry method. Finally,the gene of WSTI was cloned from wild soybean by RT-PCR and nest PCR, and its gene sequence was analyzed by bioinformatics.The drawing conclusions were as follows:(1) Compared the main ingredients of Jimo wild soybean with the cultivated soybean to determine the nutritive value of Jimo wild soybean. The results showed that the protein content, fat content and total sugar content of Jimo wild soybean were40.74%,16.91%and11.26%, respectively, which were not different from cultivated soybean, significantly. The Jimo wild soybean contained more iron, calcium and less zinc, magnesium elements than the cultivated soybean. The Jimo wild soybean contained fewer kinds of fatty acids, but much more contents of linoleic acid and linolenic acid than the cultivated soybean. The amounts of total detected eighteen kinds of amino acids in wild soybean were higher than that in the cultivated soybean. EAAI (Essential Amino Acid Index), BV (Biological Value), NI (Nutrient Index) and SRCAA (Score of Ratio Coefficient of Amino Acid) of the Jimo wild soybean were5.41%,5.0%,1.02%and3.71%higher than those of the cultivated soybean, respectively. It indicated that the Jimo wild soybean was an excellent source of high-quality protein with comprehensive and balanced composition of nutritive substance, providing the theoretical basis for further exploitation and utilization of the Jimo wild soybean.(2) The conditions for detection of soybean trypsin inhibitor were optimized by fluorescence spectrophotometry. The computational formula of the activity of WSTI was:U=(F-300.4)/1.19by the AMC standard curve. There was a certain linear relationship with the reaction speed, when the trypsin inhibitor concentration was in the range of0.1μ.g/mL~0.7μg/mL, and the detection limit was8.30ng/mL. So this method was suitable for trace detection of trypsin inhibitor during purification process.(3) The crude wild soybean trypsin inhibitor was obtained by isoelectric point precipitation and its activity value was9.86U/mg. The value was higher than that of the common variety by the same treatment. The crude inhibitor was applied to Sephadex G-50column and eluted. The second peak had trypsin inhibitory activity, with the activity value of54.69U/mg. Then peak W2was carried out on trypsin-Sepharose4B column, and only one combined peak W23displayed the activity, with value of552.85U/mg. Finally, the inhibitor was concentrated with ultrafiltration membance Pellicon5000and small molecule proteins or salt were eliminated by ultrafiltration dialysis. At last, the activity value of inhibitor was672.06U/mg. The pure powdery product WSTI was obtained by freeze-drying. After these operations, the purification fold was68.16, and the yield of activity was44.46%.(4) The WSTI was a kind of protein without sugar, which was confirmed by its maximum absorption peak at220nm and280nm through ultraviolet spectrum scan. The WSTI had high temperature and pH stability and its tolerance against DTT was strong. It also had different levels of tolerance against denaturant urea, guanidine hydrochloride, and dithiothreitol. So it was indicated that WSTI was a kind of trypsin inhibitor with high stability. The inhibition kinetics test showed that WSTI was a competitive reversible inhibitor. The inhibit constant was0.21umol/L, far lower than Km. Therefore WSTI was a kind of inhibitor with high inhibitory activity. The purity and molecular weight was determined by HPLC and two peaks were eluted. The molecular weight was9.2kDa and23.0kDa, respectively, which were mainly Bowman-Birk trypsin inhibitor with little Kunitz type. Tumor cell proliferation inhibition experiment showed that there was no significant inhibition effect on A549lung cancer cells in the0.1ug/mL~1mg/mL concentration range of WSTI.(5) The gene of WSTI was cloned from wild soybean by RT-PCR and nest PCR. The expanded product size of KSTI and BSTI were about654bp and357bp, respectively.In KSTI gene sequence, the initiation codon and termination codon were ATG and TGA, respectively. The similarity was99%between the wild soybean and cultivated soybean. It was composed of217amino acids. Relative molecular mass was24031Da and the theoretical isoelectric point was4.847. It contained26basic amino acid,31acidic amino acids,78nonpolar amino acids and82polar amino acids. Amino acid sequence contained conserved sequence of STI family, whose active site was constituted by80-Arg and90-Ser. Secondary structure was mainly composed of alpha-helical, extending chain and random curl, and the random curl proportion was the largest.In BSTI gene sequence, the initiation codon and termination codon were ATG and TAA, respectively. The similarity was99%between the wild soybean and cultivated soybean. It was composed of118amino acids. Relative molecular mass was13016Da and the theoretical isoelectric point was4.045. It contained16basic amino acids,12acidic amino acids,43nonpolar amino acids and47polar amino acids. Amino acid sequence contained conserved sequence of STI family, whose active site was constituted by62-Thr and63-Lys. Secondary structure was mainly composed of alpha-helical, extending chain and random curl, and the random curl proportion was the largest.
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