【作者】 付静；

【导师】 杨晓泉； 李理；

【作者基本信息】 华南理工大学 ， 粮食、油脂与植物蛋白工程， 2011， 博士

【摘要】 在现代化工业生产中，蛋白质水解产物比天然蛋白质具有更优越的加工性质和独特的营养特性，但是，蛋白质水解形成的肽类物质通常都具有令人难以接受的苦味，这使肽类产品在食品中的应用受到了极大的限制。雅致放射毛霉A. elegans、米曲霉A. oryzae和少孢根霉R. oligosporus分别是生产腐乳、酱油和天培的最常用生产菌种，在大豆发酵过程中，能够旺盛生长并释放出丰富的蛋白水解酶类，即肽酶酶系，并将大豆蛋白质降解为多肽和游离氨基酸，能够赋予大豆发酵食品良好的风味。本文通过对三种霉菌肽酶酶系的研究，旨在揭示其酶促降解的特点和脱苦作用的机理，以A. elegans 3.2778、A.oryzae 3042和R. oligosporus 3152为研究对象，利用不同类型的合成底物研究了霉菌内肽酶、羧肽酶、氨肽酶、二肽酶和三肽酶的种类和活力，并探讨了三种菌种产生的肽酶组成特点、其对大豆蛋白的降解特性以及脱苦作用机理。在此基础上，对具有较强脱苦作用的亮氨酸羧肽酶进行了分离纯化和性质研究。首先比较了三种菌种产生蛋白酶的条件及酶学性质，为掌握不同种类蛋白酶作用特性和进一步的研究提供理论依据。研究结果表明，三种霉菌产蛋白酶的条件不同，酶学性质也有很大差异。A. elegans产蛋白酶适宜温度为28℃，在pH 5.0⁷.0时活力最高且最稳定；A. oryzae产蛋白酶适宜温度为28³2℃，在pH 5.0⁹.0的范围内有活力，pH3.0⁴.0以及pH 6.0⁸.0的范围内稳定性强；R. oligosporus产蛋白酶适宜温度最高，为32℃，产酸性蛋白酶能力最强，在pH 3.0⁶.0范围内有较强活力，在pH 5.0⁶.0最稳定。A. elegans和A. oryzae均能产生酸性、中性及碱性蛋白酶，且A. elegans蛋白酶活力明显低于A. oryzae蛋白酶，而R. oligosporus产生大量的酸性蛋白酶和极少量中性蛋白酶。建立了实用性强的测定发酵液中羧肽酶活力的方法，即标准加入法和镉-茚三酮结合的方法，为羧肽酶定量提供了可靠手段。研究结果表明，使用纯氨基酸作为标准物制作的标准曲线计算酶活力时，得到的羧肽酶活力受样品本底值影响很大，无法客观地反映羧肽酶活力高低。本研究发现，采用加入适当稀释的粗酶液的标准加入法可以有效地消除样品本身带来的测定误差，并与纯氨基酸标准曲线（A）得到的酶活力值之间差异非常显著（ P <0.05 ）。当酶液稀释合适的倍数，加入灭活粗酶液制备的标准曲线B和加入相同稀释倍数的活性粗酶液标准曲线C得到的酶活力值之间没有显著差异（ P >0.05 ）。利用多种类型的合成底物肽，系统地研究了三种霉菌外肽酶的底物特异性，研究发现，三种霉菌均可产生丰富的羧肽酶、氨肽酶、二肽酶和三肽酶，但不同霉菌外肽酶的底物特异性不同。A. elegans和R. oligosporus的羧肽酶在中性条件下对羧基末端和次末端为Leu、Phe和Tyr的肽段有较强的水解能力，其中Z-Phe-Leu是最适底物，在酸性条件下的催化能力则明显下降；A. oryzae羧肽酶在酸性条件下对羧基末端是疏水性氨基酸（如Tyr、Leu和Phe）和次末端为Glu的底物的水解催化能力很强，其中Z-Glu-Tyr是最适底物；三种霉菌在中性条件下苯丙氨酸氨肽酶、亮氨酸氨肽酶的活力都很高，尤其是R. oligosporus所产生的亮氨酸氨肽酶活力最高；氨肽酶在pH4.0处均无活力；三种霉菌都具有很强的二肽酶和三肽酶活力，A. oryzae的二肽酶和三肽酶在pH4.0时作用范围最广，能够催化所有检测底物降解，特别是催化Leu-Tyr降解的能力比较强，A. elegans的二肽酶和三肽酶在pH7.0的活力比A. oryzae更高，也能够催化所有检测底物的降解，以Gly-Leu-Phe和Tyr-Phe为底物时活力最高，R. oligosporus的二肽酶和三肽酶在pH7.0时的活力比A. elegans还要高，以Trp-Leu和Gly-Leu-Phe为底物时活力最高，但不能降解Leu-Tyr。研究和比较了在不同pH条件下，三种霉菌蛋白质水解酶提取液对大豆分离蛋白（ SPI ）的酶促降解作用以及对苦味肽的脱苦作用。水解作用的研究结果表明，霉菌外肽酶能够显著降低苦味肽的苦味，特别是在酸性条件下，三种霉菌所产生的蛋白质水解酶对SPI的降解能力都较强，能够在3.0小时内将SPI的7S组分和11S组分完全降解，水解产物均没有苦味，尤其是A. elegans和R. oligosporus催化SPI降解后生成的游离氨基含量较少，表明这两种霉菌在水解蛋白的制备方面有良好的应用前景。脱苦作用的研究结果表明，苦味肽在经过霉菌外肽酶的处理后，苦味显著下降，特别是在酸性条件下，在没有氨肽酶活力存在时，脱苦作用仍然很强，而且A. elegans和R. oligosporus的蛋白酶提取液处理过的水解液中游离氨基酸生成量很少，其中73%是疏水性氨基酸，表明A.elegans和R. oligosporus羧肽酶是脱苦关键酶。研究了固态发酵A. elegans 3.2778产亮氨酸羧肽酶，主要考察亮氨酸羧肽酶的产酶条件、纯化过程以及酶学性质。发现该菌株产亮氨酸羧肽酶的适宜条件为25°C培养60h。亮氨酸羧肽酶最适作用pH 6.0⁷.2，最适温度40°C⁴5°C，在30°⁴0°C之间具有良好的温度稳定性。Mg²⁺能够显著提高亮氨酸羧肽酶活力，Cu²⁺、Fe²⁺、Ni²⁺、Hg²⁺对亮氨酸羧肽酶的活力具有强烈抑制作用，1，10-邻菲啰啉、苯甲基磺酰氟和二异丙基磷酰氟可以完全抑制亮氨酸羧肽酶活性。更多还原

【Abstract】 Proteolytic enzyme treatment has been commonly used to improve the process,functional, and nutritional properties of food proteins. However, a prevailing problem in theiruse has been that many proteins yielded bitter-tasting peptides during the hydrolysis process.A. elegans, A. oryzae and R. oligosporus are widely applied in the soybean fermentationindustry, they all have powerful ability to secret many kinds of peptidase, and contributeexcellent flavor to the final products. The aim of this study was to explore endopeptidase andexopeptidase of three mold species frequently utilized in soybean products, i.e. A. elegans, A.oryzae and R. oligosporus, determine their component characteristics via different kinds ofartificially synthesized substrates, and analyze the key enzymes with debittering potency onAlcalse soybean hydrolysates at different pHs. Main results are as follows:The culture condition and the properties of proteases from A. elegans, A. oryzae, and R.oligosporus and the properties of the protease system were compared firstly in this research.The culture condition and properties of proteases from different molds showed distinctivecharacteristics: The protease system from A. elegans consist of acid, neutral and alkalineprotease, but has much lower activity than which from A. oryzae, the yield of acid protease isrelatively higher in neutral and slightly acid media, but the yield of neutral and alkalineprotease is nearly the same when cultured in acid and basic media at 28oC, the proteasesystem from A. elegans is active in the range of pH 5.0⁹.0, stable in pH5.0⁷.0 and the mostactive in pH 5.0⁶.0. The protease system from A. oryzae consist of acid, neutral and alkalineprotease, and has the highest activity among the proteases system from three molds, the yieldof acid protease, neutral protease and alkaline protease from A. oryzae is higher when culturedin acid, neutral and alkaline medium at 28³2oC respectively, the protease system of A.oryzae has fairly strong activities in the range of pH 5.0⁹.0, and is stable between pH3.0⁴.0 and pH 6.0⁸.0; the protease system from R. oligosporus mainly consists of acidprotease with highest level, the enzyme yield is relatively higher when cultured in acidmedium of pH2.5⁴.0, at 32oC, the enzyme system is most active among pH 3.0 6.0 andstable around pH5.0⁶.0.Carboxypeptidase activity from A. elegans bran koji was investigated via absorbance at507 nm after being stained by Cd-nihydrin solution, with calibration curve A, which wasmade by a set of known concentration standard leucine （mmol/L）, calibration B, made bythree sets of known concentration standard leucine solutions （mmol/L） with the addition ofthree concentrations inactive crude enzyme extracts, and calibration C, made by three sets of known concentration standard leucine solutions （mmol/L） with the addition of threeconcentrations crude enzyme extracts. The results indicated that both the calibration curve Band the calibration curve C were capable to meet the demand of precision and sensitivity ofcarboxypeptidase activity determination for there was no difference in significant enzymeactivity between the calibration B and calibration C if the proper dilute multiple was used,while the enzyme activities were always overestimated when calibration curve A was used,even though the inactive crude enzyme extract was intentionally chosen as the control. It wasconcluded that the addition of crude enzyme extracts to the calibration was necessary toeliminate the interference of free amino acids and related compounds presented in crudeenzyme extract.The characteristics of exopeptidase from the 3 molds on different kinds of syntheticsubstrates were also explored. The result indicated that carboxypeptidases in A. elegans and R.oligosporus were of much higher activity under neutral condition than acidic condition, and ofgreat preference to the hydrophobic synthetic substrates with Leu, Phe and Tyr at C-terminal,and Z-Phe-Leu was their commol/Lon best substrate; however, carboxypeptidase in A. oryzaewere of much higher activity on substrates under acidic condition than under neutral condition,and its best substrate was Z-Glu-Tyr. The aminopeptidases, dipeptidyl-peptidase, dipeptidasesand tripeptidase in A. elegans and R. oligosporus all had a distinct pH preference to neutralcondition than acidic condition. Leu-ρNA, Gly-Leu-Phe both were the best substrate ofaminopeptidase and tripeptidase in A. elegans and R. oligosporus respectively. Leucineaminopeptidase activity in A. elegans was almost the same as that from A. oryzae, and bothwere at least 2.5 times lower than leucine aminopeptidase in R. oligosporus. However,dipeptidase from R. oligosporus had no power to catalyze the substrate Leu-Tyr, but showedhighest enzyme activity for substrat Trp-Leu.Proteolysis of soy protein isolates（SPI） at different pHs by crude extracts in A. elegans, R.oligosporus and A. oryzae was investigated. Under acidic condition,β-conglycinin faction andglycinin faction both were easily hydrolyzed within three hours by three mold extracts, at thesame time, amount of free amino acids, mainly hydrophobic amino acids, such as leucine,phenylalanine and tyrosine, released much less than which under neutral and alkali condition.A. elegans expressed preference to basic units of glycinin under alkalic condition. Nobitterness was produced during each hydrolysis process.Bitter pepitde solution, obtained by hydrolyzing soy protein isolates with Alcalase, wastreated with extracts from A. elegans, R. oligosporus and A. oryzae. These three molds wereall efficient tools to decrease the bitterness of bitter peptides, because of their powerful exopeptidase activities. A. elegans and R. oligosporus peptidases liberated least free aminoacid （consisted of 73% hydrophobic amino acid） under acidic condition, displaying goodapplication prospect in protein hydrolyzate industry.Studies on leucine carboxypeptidase characteristics of extract solution produced by A.elegans 3.2778 in solid state fermentation of wheat bran were conducted, with the syntheticsubstrate N-terminal blocked peptide Z-Phe-Leu and Cd-ninhydrin method. The enzyme yieldis relatively higher when cultured under 32oC for 60h. Leucine carboxypeptidase activity wasobserved at pH 6.6 and 40 oC respectively. The enzyme actively exists between pH 5.0 and 9.5but unstable above 45 oC. It was activated by Mg²⁺ at 0.99 mmol/L and 9.10mmol/L, however,partially inhibited by Ca²⁺, Zn²⁺, Ba²⁺ and Mn²⁺ at two concentrations. Leucinecarboxypeptidase activity was completely inhibited by 1,10- phenanthroline, as well as PMSFand DFP.更多还原