發(fā)布時間：2018-06-16 02:46

  本文選題：模擬酶切 + 超聲波��； 參考：《江蘇大學(xué)》2015年博士論文

【摘要】：玉米蛋白是濕法生產(chǎn)玉米淀粉的副產(chǎn)物,約含70%的蛋白質(zhì),其獨(dú)特的理化組成以及必需氨基酸的缺乏嚴(yán)重限制了其在食品工業(yè)中的應(yīng)用。本文旨在通過酶法水解以提高玉米蛋白的生物利用度,通過玉米蛋白的超聲波預(yù)處理提高其酶解效率和產(chǎn)物得率。本文開發(fā)了基于生物信息學(xué)平臺的蛋白質(zhì)酶切過程計算機(jī)模擬軟件,利用該軟件完成蛋白酶的自動篩選；根據(jù)篩酶結(jié)果,研究了不同酶解程度(水解度)下玉米蛋白生物利用度的變化規(guī)律及其分子機(jī)制；針對玉米蛋白存在因富含疏水性氨基酸而導(dǎo)致的溶解性低、結(jié)構(gòu)致密這一分子結(jié)構(gòu)缺陷,研究了雙頻功率超聲波(聚能式、發(fā)散式)預(yù)處理對玉米蛋白酶解特性的影響；通過紫外光譜、熒光光譜、傅里葉變換紅外光譜、圓二色譜、掃描電鏡和原子力顯微鏡等多尺度分析方法和測試手段研究了雙頻功率超聲波促進(jìn)玉米蛋白酶解的作用機(jī)理；根據(jù)超聲的作用機(jī)制和玉米蛋白的分子結(jié)構(gòu)特征,采用米氏方程、一級動力學(xué)模型、阿倫尼烏斯方程以及過渡態(tài)理論研究了發(fā)散式雙頻掃頻超聲預(yù)處理對玉米蛋白酶解反應(yīng)動、熱力學(xué)參數(shù)的影響。本文得出以下主要研究結(jié)論：(1)開發(fā)出了可以滿足蛋白質(zhì)酶切基本過程模擬與結(jié)果分析的計算機(jī)軟件,該軟件能對蛋白質(zhì)進(jìn)行單(雙)酶定向靶位酶切,能以指定的蛋白水解度為特征指標(biāo)完成蛋白酶的自動篩選,并可以對酶切產(chǎn)物的分子量分布及其端點(diǎn)疏水性氨基酸的含量進(jìn)行統(tǒng)計分析。(2)酶解反應(yīng)顯著提高了玉米蛋白的生物利用度,且提高幅度與蛋白水解度直接相關(guān)。體外模擬消化表明,水解度通過影響酶解液的多肽分子量分布和氨基酸組成來影響其生物利用度。酶解液中分子量大小為200-500 Da組分的含量幾乎不受胃蛋白酶、胰蛋白酶的影響。隨水解度的增大,游離疏水性氨基酸尤其Ala、Met、Ile、Val和Phe的含量增加,Thr和Leu含量減小；酶解液中的總氨基酸、疏水性氨基酸尤其是Leu、Pro和Ala含量隨水解度的增大而增加。氨基酸消化率表明,酶解液中疏水性氨基酸Met、Ile、Pro、Ala的消化率顯著下降,Thr的消化率顯著上升,水解度對親水性、疏水性氨基酸的平均消化率無顯著性影響。(3)針對蛋白質(zhì)的超聲預(yù)處理,以水解度和蛋白轉(zhuǎn)化率為指標(biāo),分別優(yōu)化得出了聚能式和發(fā)散式兩種功率超聲波的最佳工作模式及其工作參數(shù)。與無超聲的常規(guī)酶解相比,玉米蛋白經(jīng)聚能順序雙頻超聲預(yù)處理后,其水解度和蛋白轉(zhuǎn)化率分別提高了45.3%和59.3%；經(jīng)發(fā)散雙頻掃頻超聲預(yù)處理后,其水解度和蛋白轉(zhuǎn)化率分別提高了38.8%和54.7%。玉米蛋白經(jīng)超聲尤其是聚能順序雙頻超聲預(yù)處理后,其酶解液的分子量分布范圍更窄,主要集中在200-1000 Da之間。(4)紫外光譜、熒光發(fā)射/激發(fā)光譜、表面疏水性結(jié)果表明,超聲預(yù)處理能暴露出埋藏在分子內(nèi)部的疏水性內(nèi)核、使得蛋白質(zhì)分子舒展,有利于酶解反應(yīng)的進(jìn)行。傅里葉變換紅外光譜和圓二色譜分析結(jié)果表明,經(jīng)超聲預(yù)處理后,蛋白各二級結(jié)構(gòu)(a-螺旋、β-折疊、β-轉(zhuǎn)角和無規(guī)則卷曲)的相對含量發(fā)生了變化,且超聲對玉米蛋白中的谷蛋白的作用強(qiáng)于對醇溶蛋白的作用。掃描電鏡結(jié)果表明,盡管順序雙頻處理后的醇溶蛋白出現(xiàn)了一定的聚集,但超聲預(yù)處理能破壞蛋白的微觀結(jié)構(gòu)、減小顆粒的尺寸,增大酶與蛋白質(zhì)的接觸機(jī)會。亞顯微結(jié)構(gòu)及納米機(jī)械力學(xué)特性分析表明,超聲預(yù)處理能減小谷蛋白的顆粒高度和表面粗糙度,能減小醇溶蛋白的楊氏模量和剛度,增加蛋白的粘附力。發(fā)散式雙頻掃頻超聲適合于疏水性較強(qiáng)的蛋白預(yù)處理,而聚能式順序雙頻超聲適合于疏水性較弱的蛋白預(yù)處理。(5)動力學(xué)研究表明,玉米蛋白經(jīng)雙頻掃頻超聲預(yù)處理后,其酶解反應(yīng)的表觀常數(shù)(KM值)降低了26.1%、表觀崩解常數(shù)(kA值)增加了7.3%,說明超聲處理后的玉米蛋白與酶的親和力和結(jié)合頻率得到提高；熱力學(xué)分析表明,玉米蛋白經(jīng)雙頻掃頻超聲預(yù)處理后,在293、303、313和323 K的反應(yīng)溫度下的酶解反應(yīng)速率常數(shù)(k)分別提高了84.8%、41.9%、28.9%和18.8%,酶解反應(yīng)的活化能(Ea)、活化焓(ΔH)、活化熵(△S)分別降低了23.0%、24.3%和25.3%,意味著酶解反應(yīng)更容易進(jìn)行。
[Abstract]:Corn protein is a by-product of corn starch production by wet method. It contains about 70% protein. Its unique physical and chemical composition and the lack of essential amino acids seriously restrict its application in the food industry. This paper aims to improve the bioavailability of corn protein by enzymatic hydrolysis and improve its enzymatic hydrolysis through ultrasonic pretreatment of corn protein. Efficiency and product yield. In this paper, a computer simulation software based on bioinformatics platform was developed. The software was used to screen the protease automatically. According to the results of the enzyme, the changes of the bioavailability of corn protein and its molecular mechanism under different degree of hydrolysis (degree of hydrolysis) were studied. There is a structural defect of low solubility and compact structure caused by rich hydrophobic amino acids. The effect of dual frequency power ultrasound (concentrated energy, divergent) pretreatment on the hydrolysis of corn protease is studied. Ultraviolet spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular two chromatography, scanning electron microscopy and atomic force microscopy are used. The mechanism of dual frequency power ultrasound promoting the hydrolysis of corn protease is studied by multiscale analysis and testing. According to the mechanism of ultrasound and the molecular structure of corn protein, the Michaelis equation, the first order kinetic model, the Arrhenius equation and the over state theory are used to study the divergent dual frequency frequency sweep ultrasound. The effect of pretreatment on enzymatic hydrolysis of corn protease and the influence of thermodynamic parameters. This paper draws the following main conclusions: (1) a computer software can be developed to simulate the basic process simulation and result analysis of protein enzyme cutting. The software can cut the protein to target enzyme with a single (double) enzyme and can be characterized by the specified degree of protein hydrolysis. The index completes the automatic screening of protease, and can analyze the molecular weight distribution of the enzyme cut products and the content of the hydrophobic amino acids in the endpoint. (2) the enzymatic hydrolysis reaction significantly improves the bioavailability of the corn protein, and the increase in the amplitude is directly related to the degree of protein hydrolysis. The molecular weight distribution and amino acid composition of the polypeptide affect its bioavailability. The content of the molecular weight of 200-500 Da in the hydrolysate is almost unaffected by pepsin and trypsin. With the increase of the degree of hydrolysis, the content of free hydrophobic amino acids especially Ala, Met, Ile, Val and Phe is increased, the content of Thr and Leu is reduced; the content of Thr and Leu is reduced; the content of Thr and Leu is reduced; the content of Thr and Leu is reduced; The content of total amino acids, hydrophobic amino acids, especially Leu, Pro and Ala increased with the increase of the degree of hydrolysis. The digestibility of the amino acid digestibility in the hydrolysate showed that the digestibility of Met, Ile, Pro, Ala decreased significantly, the digestibility of Thr increased significantly, and the degree of hydrolysis had no significant influence on the average digestibility of hydrophobicity and hydrophobic amino acids. (3) needle The optimum working mode and its working parameters of two kinds of power ultrasound were optimized, including the degree of hydrolysis and the conversion rate of protein. Compared with the conventional enzymatic hydrolysis without ultrasonic, the hydrolysis degree and the protein conversion rate of corn protein were respectively compared with that of the two frequency ultrasonic pretreatment. After 45.3% and 59.3% higher, the hydrolysis degree and protein conversion rate increased by 38.8% and 54.7%. corn protein after ultrasonic pretreatment, and the molecular weight distribution of the hydrolysate was narrower, mainly between 200-1000 Da. (4) ultraviolet spectrum, fluorescence emission / excitation. The results of spectrum and surface hydrophobicity show that ultrasonic pretreatment can expose the hydrophobic core that is buried inside the molecule, which makes the protein molecules stretch and facilitate the enzymatic reaction. Fourier transform infrared spectroscopy and circular two chromatographic analysis show that after ultrasound pretreatment, the two structure of protein (a- spiral, beta fold, beta corner and) The relative content of irregular curling changed, and the effect of ultrasound on glutenin in corn protein was stronger than that of gliadin. The results of scanning electron microscopy showed that although a certain concentration of gliadin appeared after the sequence double frequency treatment, the ultrasonic pretreatment could destroy the microstructure of the protein, reduce the size of the particles, and increase the enzyme. The analysis of the microstructure and mechanical mechanical properties of the protein shows that the ultrasonic pretreatment can reduce the grain height and the surface roughness of the protein, reduce the young's modulus and stiffness of the gliadin, and increase the adhesion of the protein. The energetic sequence dual frequency ultrasound is suitable for weak hydrophobic protein pretreatment. (5) kinetic studies show that the apparent constant (KM value) of the enzymolysis reaction of corn protein decreased by 26.1% and the apparent disintegration constant (kA value) increased by 7.3%, indicating the affinity and binding frequency of the corn protein with the enzyme after ultrasonic treatment. The thermodynamic analysis showed that the enzyme hydrolysis rate constant (k) increased by 84.8%, 41.9%, 28.9% and 18.8% at the reaction temperature of 293303313 and 323 K, and the activation energy (Ea), activation enthalpy (delta H), and the activation entropy (delta S) decreased by 23%, 24.3% and 25.3% respectively. The enzymolysis reaction is more easily carried out.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：TS239;TQ936.2

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