沙蒿胶磁性微球固定化脂肪酶及部分理化性质的研究

以沙蒿多糖-壳聚糖复合磁性微球为载体,采用物理吸附法固定化脂肪酶,对固定化过程中对酶活力有影响的各种因素做了研究,同时对固定化酶的部分理化性质、最适pH、最适温度、酶的热稳定性以及表现米氏常数与游离酶做了比较.固定化酶的Km小于游离酶的Km,其最适pH和最适温度分别为8.0和50℃,而且固定化脂肪酶具有良好的热稳定性、可应用性和重复使用性.     

PVA-SA-活性炭共聚物固定脂肪酶的研究

以聚乙烯醇(PVA)-海藻酸钠(SA)-活性炭共聚物为载体,对脂肪酶进行固定化.研究了活性炭浓度、酶初始浓度、缓冲液pH值、吸附时间及温度对固定化酶的活性及蛋白载量的影响.结果表明:在聚乙烯醇(PVA)-海藻酸钠凝胶中加入1 g/dL活性炭制成复合凝胶球,在25 ℃、pH值5.5的条件下对30 mg/mL的酶液吸附12 h,所得吸附固定化脂肪酶的活性较好.吸附固定的脂肪酶较游离酶的最适作用温度升高10 ℃,最适作用pH范围变宽,且向碱方向偏移0.2个单位.     

SDS-Chitin-PAGE电泳法分析大葱叶几丁质酶特性

使用Sodium dodecyl sulfate-几丁质-聚丙烯胺酰胺凝胶电泳(SDS-Chitin-PAGE)方法分析了大葱葱叶几丁质酶的特性.在大葱叶组织中共检测到三种几丁质酶同工酶,分别命名为CH-A、CH-B、CH-C.分析结果表明,这三种几丁质酶具有SDS抗性,β-巯基乙醇对其活性也无显著影响.在SDS-Chitin-PAGE前,样品缓冲溶液与样品蛋白粗提液孵化处理的最适温度为24℃、时间为10～20min.凝胶电泳酶活分析表明,大葱葱叶中CH-A、CH-B的耐热温度可达60℃,CH-C的耐热温度达到70℃.葱叶中CH-A的最适pH为5.2～5.6,CH-B的最适pH为4.0～4.4,CH-C的最适pH为6.6～8.0.     

磷脂酶A_1(Lecitase Ultra)的氨基酸序列分析及催化机理

研究磷脂酶A_1(Lecitase Ultra)的纯化、酶蛋白的氨基酸序列及活性中心的氨基酸残基组成,并推导水解磷脂酰胆碱(Pc)的机理。超滤法和RP-HPLC纯化酶液,除去了山梨醇和山梨醇盐,纯度97.7%;基质辅助激光解吸电离飞行时间质谱(MALDI SYNAPT Q-TOF MS)和二级质谱(MS/MS)对酶蛋白进行分析,确定了氨基酸序列组成;数据库(http://www.matrixscience.com)比对,发现氨基酸序列信息与棉状嗜热丝孢菌(T.lanuginosus)脂肪酶和尖孢镰刀菌(F.oxysporum)脂肪酶高度吻合,序列中1-284是T.lanuginosus脂肪酶的氨基酸序列,序列中285-339是F.oxysporum脂肪酶的氨基酸序列;Lecitase Ultra和Sn-1-C16:0/sn-2-C2:0-PC的分子对接证实了酶活中心的三联体"Ser-His-Asp"催化机理。     

不同大麦籽粒品质及发芽后蛋白质含量和主要水解酶活力变化的研究

研究了12种国内外大麦籽粒的外观和基本理化品质,检测其发芽前后清蛋白、球蛋白、醇溶蛋白和谷蛋白4种蛋白质亚组分的含量及主要水解酶活力的变化,探讨了部分蛋白亚组分与总蛋白、酶与蛋白及酶与酶之间的相关性.结果表明:1号和3号大麦籽粒品质较好,具有开发啤酒大麦的潜力;发芽后大麦总蛋白含量基本不变,清蛋白大幅增加,球蛋白略降低,贮存蛋白(醇溶蛋白与谷蛋白)总体降低.相关性分析表明:大麦和麦芽中贮存蛋白的含量反映了其总蛋白的含量;α-淀粉酶与清蛋白呈负相关性,蛋白酶与清蛋白呈正相关性,β-淀粉酶与总蛋白、蛋白亚组分及蛋白酶之间存在显著正相关性.     

纤维素酶热稳定机制的计算分析

对水解糖苷酶第45家族的纤维素酶系进行计算分析,计算结果表明,与此家族酶系的热稳定性呈负相关的氨基酸对为:丙氧酸-谷氨酸(AE)、苯丙氨酸-赖氨酸(FK)和丙氨酸-异亮氨酸(AI);呈正相关的氨基酸对:甲硫氨酸-脯氨酸(MP)和甲硫氨酸-甘氨酸(MG),并引入氨基酸热稳定常量计算验证其相关性,发现呈负相关的氨基酸对均为显著相关,而呈正相关的氨基酸对显著性不明显.这个结果可以为此家族纤维素酶的基因工程改造提供一定参考.     

脂肪酶的基因克隆、序列分析、高效表达及其催化特性

分别以R. miehei 3.4960基因组和总RNA为模板,通过PCR和RT-PCR扩增得到脂肪酶全长基因和脂肪酶cDNA碱基序列,两碱基序列测序结果比对表明：脂肪酶DNA碱基序列由5个内含子和6个外显子组成,5个内含子大小分别为75、58、64、73、59bp,位于DNA序列(以起始密码子ATG的碱基A为1)的600～674位、929～986位、1076～1139位、1227～1299位、1382～1440位碱基处。脂肪酶RML mRNA碱基序列与已公布的R. miehei脂肪酶基因mRNA(EMBL Nucleotide Sequence Database Accession No. A02536)碱基序列有5个碱基的差异,其编码的氨基酸序列有1个发生了改变。利用酿酒酵母表达质粒pICAS1,实现了脂肪酶RML基因在酿酒酵母中的高效分泌表达,并对重组脂肪酶RML进行了相关酶学性质分析。酶学性质研究表明：重组酶的最适反应温度为45℃,最适pH值为8.6;该酶偏爱中链长的酯(C8～C12),对12个碳的酯具有最佳活性,对C16长链酯也有较高的活性,对短链酯的活性较弱;金属离子Cu2+、Fe2+对酶活性有显著抑制作用,Ca2+和Mg2+对酶活有部激活作用。     

嗜冷菌对牛乳货架期的影响

采用Azocasein法和碱式滴定法对嗜冷茵产蛋白酶和脂肪酶的活力进行了测定.研究了嗜冷茵在原料乳中的生长规律.结果表明,嗜冷茵产蛋白酶和脂肪酶的最佳pH值为7.0,最适培养温度在30℃,产酶高峰分别出现在30 h和24 h,在最适培养条件下嗜冷茵数的动态增加与蛋白酶和脂肪酶活力变化趋势大致相同.原料乳在低温贮藏过程中,嗜冷茵数与蛋白质、脂肪、水分、密度、pH值也存在有数学相关性.     

超声波对脂肪酶酶学特性的影响

研究了超声波对脂肪酶水解橄榄油反应的影响及超声波处理后脂肪酶的酶学特性.研究结果表明:超声波促进脂肪酶水解橄榄油反应的最佳条件是:超声功率200 W、超声频率20 kHz、超声处理时间8 min.在最佳超声条件下,脂肪酶的活力有所提高,但其最适作用pH和最适温度没有改变.     

UV-C辐照对凡纳滨对虾虾头主要内源酶酶学性质的影响

以凡纳滨对虾虾头为研究对象,探究UV-C辐照对其主要内源酶的影响。用功率为30 W的UV-C紫外灯以20 cm高度、辐照20 min,处理虾头均浆液,检测内源酶酶活,分析比较辐照前后温度、pH对其内源酶酶活的影响规律。从虾头中检测出酸性及碱性蛋白酶、脂肪酶、几丁质酶和多酚氧化酶等酶活性; UV-C辐照前后,脂肪酶、几丁质酶的最适pH有所变化,脂肪酶最适pH由10变为6～10,几丁质酶最适pH由5变为6左右;各主要内源酶最适温度无明显变化; UV-C辐照后,在最适条件下酸性蛋白酶酶活升高23. 99%,碱性蛋白酶升高37. 49%,脂肪酶升高21. 37%,几丁质酶升高37. 70%,多酚氧化酶升高46. 52%。结果显示,UV-C辐照对凡纳滨对虾虾头酸性及碱性蛋白酶、脂肪酶、几丁质酶和多酚氧化酶等主要内源酶均有激活作用。     

含酪氨酸二肽在脂质体体系中的抗氧化活性与供电子能力

采用紫外波段扫描对5 种含酪氨酸二肽（酪氨酸-酪氨酸、酪氨酸-苯丙氨酸、苯丙氨酸-酪氨酸、酪氨酸-丙氨酸和酪氨酸-丝氨酸）的紫外吸收特性进行检测，并采用还原力法、循环伏安法及2,2-偶氮二(2-甲基丙基咪)二盐酸盐诱导氧化脂质体模型对其抗氧化活性进行评价及比较，以探究影响5 种含酪氨酸二肽抗氧化活性的因素及其在均相与非均相体系中的活性差异与联系。结果表明：构成含酪氨酸二肽的氨基酸组成及排列顺序对于二肽的紫外吸收特征、供电子能力以及脂质氧化抑制率有显著影响，其中酚羟基的数量对其抗氧化活性起决定性作用。与酪氨酸-苯丙氨酸相比，苯丙氨酸-酪氨酸在3 种抗氧化活性评价方法中均显示出更高的活性。在脂质体体系中，含酪氨酸二肽的脂质氧化抑制率除了与其供电子能力显著相关（P＜0.05）外，还与其自身的亲脂或亲水性具有一定的相关性。     

一种新的氨基酸描述符SVHEHS在生物活性肽QSAR中的应用研究

对20种氨基酸的457种性质参数按疏水性质、电性特征、氢键贡献和立体特征进行分类后,并各自进行主成分分析(PCA),得到一种新的氨基酸结构描述符SVHEHS(score vector of hydrophilicity, electronic, hydrogen bond contribution and steric properties)。用该描述符分别对一系列血管紧张素转化酶抑制二肽以及苦味二肽进行序列表征,并用来与生物活性建立多元线性回归模型。血管紧张素转化酶抑制二肽、苦味二肽模型的相关系数、交叉验证相关系数、均方根误差分别为0.936、0.854、0.259和0.949、0.886、0.136,同时还对所得模型进行了外部验证。结果表明,该描述符建立的模型具有较好的拟合与预测能力,用于生物活性肽的定量构效关系研究是理想的。     

Novel diketopiperazine metabolism in a microorganism: two-step hydrolysis of cyclo(Gly-Leu) to amino acids and preliminary characterization of cyclo(Gly-Leu) hydrolase and dipeptidase

A bacterium, strain NM 5-3, isolated from soil exhibited the highest cyclo(Gly-Leu) (CGL)-hydrolyzing activity and was identified as Agrobacterium radiobacter. The reaction products from CGL were dipeptides (Leu-Gly and Gly-Leu) and amino acids (Leu and Gly). Inhibitors for the dipeptidase of this strain did not inhibit the hydrolysis of CGL to dipeptides, indicating that two distinct enzymes, CGLase and a dipeptidase, were involved in its hydrolysis. The activities of these two enzymes were separated by anion-exchange column chromatography. The results indicated that strain NM5-3 hydrolyzed CGL via the dipeptides to the corresponding amino acids. The CGLase fraction was found to catalyze the hydrolysis of cyclo(Gly-D-Leu), cyclo(Gly-Gly), cyclo(L-Ala-Gly), and cyclo(D-Ala-Gly). On the other hand, the dipeptidase fraction exhibited L-specific substrate specificity.     

PYROGLUTAMYL DIPEPTIDES IN MUSHROOM, AGARICUS CAMPESTRIS

SUMMARY: A small fraction, containing evidence for the presence of pyroglutamyl dipeptides and an N-pyroglutamylhexosamine. was isolated from the edible, commercial mushroom. Agaricus campestris. Upon hydrolysis, this isolate liberated a copious amount of glutamic acid, together with smaller quantities of other amino acids and a hexosamine. Analyses of the unhydrolyzed and hydrolyzed portions of the fraction by an automatic amino acid analyzer revealed the identities and quantities of the compounds released. Additional chemical and physical methods of analysis gave supporting evidence for the presence in the mushroom in addition to proline and pyroglutamic acid of the pyroglutamyl dipeptides of threonine, aspartic acid, valine, leucine, citrulline, phenylalanine, glycine, alanine, glutamic acid and proline and also of the amino acid sugar, N-pyroglutamylglucosamine. Pyroglutamylcitrulline and N-pyroglutamylglucosamine were tentatively identified. The first 6 dipeptides are reported for the first time. A mechanism is proposed for the enzymic biosynthesis of the pyroglutamyl dipeptides in the mushroom. Results of the present work shed additional light on the complex nature of the higher basidiomycetes.     

Papain‐catalyzed synthesis of dipeptides

The cysteine proteinase papain (EC 3.4.22.2) was used to synthesize N‐protected dipeptides in a kinetically controlled reaction starting from N‐benzyloxycarbonyl‐L‐alanine methylester as the carboxyl component. Beside L‐amino acid amides, free L‐amino acids could be successfully applied to yield the corresponding Z‐dipeptides. The yields with free L‐amino acids ranged from 13–80%, obviously depending on the structure of the molecule. The evaluation of the reaction conditions for the synthesis of N‐benzyloxycarbony1‐L‐alanyl‐L‐arginine revealed that a pH of 9.5, a temperature of 25–40°C and 0.5 M L‐arginine were optimal.     

Response of forage fiber degradation by ruminal microorganisms to branched-chain volatile fatty acids,amino acids,and dipeptides

This study evaluated the effect of branched-chain volatile fatty acids (VFA; isobutyric acid, isovaleric acid), amino acids (valine, leucine), and dipeptides (valine-valine, leucine-leucine) on neutral detergent fiber (NDF) degradation by rumen microorganisms in vitro. The CP (%) and in situ NDF degradation rate (%/h) for alfalfa, bermudagrass, and pangolagrass hays, and napiergrass silage were 17.2 and 7.5, 4.7 and 3.1, 8.3 and 5.3, and 9.6 and 3.4, respectively. In vitro NDF digestibility was the lowest for bermudagrass; alfalfa and napiergrass were the highest. When the incubation contained more ammonia initially, digestibilities increased, but relative differences among forages were unchanged. Adding branched-chain VFA (2 mM) to incubations increased digestibilities more than controls on 15 out of 16 occasions. The effectiveness varied with isoacids and forages used. Amino acid (2 mM) or dipeptide (1 mM) addition consistently increased digestibility over controls. Amino acids further increased digestibility over corresponding isoacids on 14 occasions. Improvement in digestibility over control by leucine appeared to be greater than that by valine. Digestibilities with dipeptides were always greater than those with isoacids, except for one case. Dipeptide addition further increased digestibility significantly over corresponding amino acids on only six occasions, while percent improvement in digestibility numerically by dipeptides occurred in 10 cases. Valine-valine seemed to exert different effect than leucine-leucine, depending on initial ammonia availability. The results indicate that dipeptides could be more effective than isoacids and amino acids in improving NDF digestion. Forages with high CP content or rapid NDF degradation rate appeared to respond to additives to smaller degrees.     

Inhibition of dipeptidyl peptidase IV and xanthine oxidase by amino acids and dipeptides

Xanthine oxidase (XO) and dipeptidyl peptidase IV (DPP-IV) inhibition by amino acids and dipeptides was studied. Trp and Trp-containing dipeptides (Arg-Trp, Trp-Val, Val-Trp, Lys-Trp and Ile-Trp) inhibited XO. Three amino acids (Met, Leu and Trp) and eight dipeptides (Phe-Leu, Trp-Val, His-Leu, Glu-Lys, Ala-Leu, Val-Ala, Ser-Leu and Gly-Leu) inhibited DPP-IV. Trp and Trp-Val were multifunctional inhibitors of XO and DPP-IV. Lineweaver and Burk analysis showed that Trp was a non-competitive inhibitor of XO and a competitive inhibitor of DPP-IV. Molecular docking with Autodock Vina was used to better understand the interaction of the peptides with the active site of the enzyme. Because of the non-competitive inhibition observed, docking of Trp-Val to the secondary binding sites of XO and DPP-IV is required. Trp-Val was predicted to be intestinally neutral (between 25% and 75% peptide remaining after 60 min simulated intestinal digestion). These results are of significance for the reduction of reactive oxygen species (ROS) and the increase of the half-life of incretins by food-derived peptides.     

Influence of pre-cure freezing of Iberian ham on proteolytic changes throughout the ripening process

This work aimed to investigate the effect of pre-cure freezing Iberian hams on proteolysis phenomena throughout the ripening process. Non-protein nitrogen (NPN), peptide nitrogen (PN) and amino acid nitrogen (AN) as well as amino acid and dipeptide evolution followed the same trend in both refrigerated (R) and pre-cure frozen (F) Iberian hams during processing. At the different stages of ripening, there were no differences in the content of NPN and AN while F dry-cured hams had higher levels of PN than R hams at the final step. This seemed to be more related to the salt content (lower in F than in R hams) than to the pre-cure freezing treatment. Most amino acids and dipeptides detected showed higher concentrations in F than in R Iberian hams at the green stage, being rather similar at the intermediate phases. At the final stage, the effects of pre-cure freezing of Iberian hams were not well defined, higher levels of some amino acids and dipeptides were found in R than in F Iberian hams whereas other amino acids were lower in R than in F hams.     

Enzymatic conversion of cyclic dipeptides to dehydro derivatives that inhibit cell division

The cell-free extract of an albonoursin-producing strain, Streptomyces albulus KO-23, was found to catalyze the conversion of several cyclic dipeptides having Phe and aliphatic side chain-containing amino acid residues to the corresponding dehydro derivatives. 3Z-Benzylidene-6S-methyl-2,5-piperazinedione, 3Z-benzylidene-2,5-piperazinedione, and 3Z, 6Z-dibenzylidene-2,5-piperazinedione were prepared by this conversion system. Among the dehydro cyclic dipeptides prepared, tetradehydro derivatives exhibited inhibitory activity toward the first cleavage of sea urchin embryo, while didehydro derivatives did not. We previously found that cyclo(Leu-Phe) and its didehydro derivatives did not show any inhibitory activity, in contrast to high activity in the case of albonoursin. Taken together, these findings indicate that dehydrogenation at the alpha,beta-positions of both amino acid residues in this type of cyclic dipeptide is required for the inhibitory activity.     

FREE AMINO ACIDS AND DIPEPTIDES IN DRY-CURED HAM

Six types of dry-cured hams were chemically profiled for proximate composition and low molecular weight nonprotein nitrogen (NPN) fractions including free amino acids (FAA) and two dipeptides (carnosine and anserine). Results indicate that FAA were present in the six classes at different (mg/100g protein) concentrations, with Iberian and Serrano containing greater (P<0.05) amounts of most FAA. However, when analyzed as percentage (mg/100g total FAAs) values, FAA contents of Iberian and Serrano hams exhibited lower (P<0.05) tyrosine and greater (P<0.05) aspartic acid and threonine values. Within each class, large variation coefficients (>10%) for nonprotein nitrogen paralleled inconsistent values of salt content, to which NPN and FAAs were negatively correlated (P<0.01). NPN was also negatively affected by moisture in the dried muscle, indicating limited saltiness and greater muscle shrinkage as two major determinants in developing of nonprotein fractions, hence of free amino acids.