水溶性多糖酶解过程分子量变化与动力学建模

以魔芋葡甘聚糖-β-甘露聚糖酶水解体系为例,研究了水溶性多糖酶解过程中产物的分子量变化与动力学行为.利用凝胶排阻色谱法和特性粘度法,分别测定了酶解物的分子量分布和重均分子量(-Mw),结果表明:随着反应的进行,酶解物分子量分布先变宽再逐渐变窄,这是由酶在底物反应体系中的镶嵌式分布,不均一的底物分子序列结构与酶分子的选择性剪切,酶剪切的多种途径以及酶与底物的结合模式等四种因素共同作用的结果;初始阶段酶解物(-Mw)先快速下降再逐渐趋于平缓,其速率与底物浓度有关;基于酶解产物重均分子量变化规律而建立的(1/-Mw)随反应时间t变化的动力学模型,确定了常见的水溶性多糖浓度下酶解过程为零级降解,并与实验结果相当一致.     

基于酶解-膜分离集成的胶原蛋白肽分子量控制技术

采用酶解-膜分离集成工艺制备分子量范围可控的胶原蛋白多肽,研究反应过程中酶种类、截留分子量和过滤体积等因素对酶解-膜分离集成工艺的影响.结果表明,用截留分子量为3 k Da的超滤膜处理的透过液分子量主要分布在4.0,1.6和0.6k Da,比例分别为13.7%,34.8%和51.4%;用截留分子量为8 k Da的超滤膜处理的透过液分子量主要分布在8.3,4.0,1.6和0.6 k Da,比例分别为14.5%,22.7%,37.7%和25.1%;酶解-膜分离集成工艺蛋白转化速率比酶解过程提高15%,表明酶解-膜分离集成可以加快大分子蛋白转化率,并使酶解产物分子量均一可控.     

海蜇、牛骨和鳕鱼皮胶原肽的透皮吸收性能研究

以海蜇、牛骨和鳕鱼皮胶原为原料,采用酶解制备了不同分子量(1 kDa和1~3 kDa)的6种胶原肽。利用透皮扩散试验仪,考察了时间、分子量及质量浓度对胶原肽透皮吸收性的影响,通过荧光显微镜观察了异硫氰酸荧光素(FITC)标记的胶原肽对小鼠皮肤的透皮吸收过程。结果表明,6种胶原肽的质量浓度均为10 g/L时,其在24 h时透皮吸收能力由大到小依次为:1 kDa鳕鱼皮胶原肽、1 kDa牛骨胶原肽、1 kDa海蜇胶原肽、1~3 kDa海蜇胶原肽、1~3kDa牛骨胶原肽和1~3 kDa鳕鱼皮胶原肽;1 kDa胶原肽透皮吸收能力优于1~3 kDa胶原肽。牛骨和海蜇胶原肽的质量浓度越大,透皮吸收性越好,而鳕鱼皮胶原肽则相反。3种来源胶原肽单位面积累积透过量均随时间延长而增加,分子量越大透皮吸收性能越差。     

低聚木糖生产用木聚糖酶的选择性合成

以里氏木霉(Trichoderma reesei)Rut C-30为产酶菌,研究了碳源、碳氮比对木聚糖酶酶系组成的影响.低分子量组分较多的木聚糖有利于促进内切-β-木聚糖酶的合成,酶解产物中低聚木糖的含量较高(80.70%).低碳氮比有利于促进内切-β-木聚糖酶的合成,抑制外切-β-木糖苷酶的合成.以低分子量较多的木聚糖(7g/L)为碳源,降低培养基的碳氮比为4.0,调控培养60h,用该木聚糖酶酶解粗木聚糖,产物中低聚木糖占总糖的86.32%.     

大豆蛋白酶解产物的分子量分布与其发泡性能关系的研究

研究木瓜蛋白酶水解大豆蛋白所得到的酶解产物的分子量分布与其发泡性能的关系。酶解产物经过分离纯化,根据标准曲线的回归方程得到,大豆多肽的分子量分别为大于5000 Da和1148 Da、501 Da三个级分,其中第二个为主要成分。通过泡沫性能测试,结果表明,分子量在2000 Da以下的酶解产物的发泡性能最好,依次为分子量在3000~5000 Da之间的产物、2000~3000 Da之间的、5000~10000 Da之间的,分子量10000 Da以上的产物的发泡性能最差。     

南极菌Pseudoalteromonas sp.A211-5产碱性α-淀粉 酶Amy172的克隆表达及酶学性质研究

对具有淀粉降解活性的南极菌Pseudoalteromonas sp.A211-5进行测序分析,根据基因注释结果,获得了一条基因序列为2 139 bp的淀粉酶基因,命名为amy172;通过特异性引物克隆获得Amy172的基因序列并进行异源表达;采用Ni-NTA层析柱对重组α-淀粉酶Amy172进行纯化,采用DNS法测定其酶学性质,采用薄层层析(TLC)技术对其酶解产物进行分析。结果表明:SDS-PAGE显示在分子量为79 kDa左右有一条明显的目的蛋白条带;重组α-淀粉酶Amy172最适温度为50℃,最适pH值为10.0,且在pH值5.0～10.0的范围内仍能保持80%以上的酶活,金属离子Fe~(3+)、Mn~(2+)、Mg~(2+)、Fe~(2+)、Ca~(2+)、K~+、Sr~(2+)、Ni~(2+)和Ba~(2+)对酶活性具有抑制作用;TLC分析Amy172的酶解产物为葡萄糖、麦芽糖、麦芽三糖和麦芽四糖。     

凝胶过滤色谱法研究酶解过程中木聚糖分子结构的变化

采用凝胶过滤色谱法研究了木聚糖在酶解过程中分子量分布的变化。结果表明,木聚糖酶对高分子量组分的木聚糖的降解能力较差,而主要降解分子量介于15000～3000之间组分的木聚糖,使得这部分木聚糖变为分子量更低的木聚糖组分。随着酶解时间的延长,酶解得率上升,酶解产物中低聚木糖含量增多,未被酶解的木聚糖的平均聚合度上升,当酶解时间超过4h时,这种变化趋势缓慢。随着酶解轮次的增加,酶解产物中可溶性木聚糖的含量越来越少,木聚糖被降解的难度越来越大。在实际生产中,用来作为低聚木糖生产的木聚糖其聚合度应在20～100之间,酶解时间以4h为宜,重复利用未水解木聚糖的轮次以2轮为宜。     

利用RP-HPLC-MS测定绿豆中多肽的技术研究

本实验对绿豆分离蛋白酶解产物的研制工艺条件、产物的理化性质及分子量分布进行了全面深入的研究。通过利用反相高效液相色谱-质谱联用(RP-HPLC-MS)技术针对该体系建立了可靠、有效的液相色谱分离方法,及质谱分析方法,对酶解产物(绿豆肽)进行了分离分析。结果显示:绿豆分离蛋白经酶解后得到的肽混合物分子量基本都在1000 Da以下,本实验所得到的绿豆分离蛋白酶解产物适合应用于食品及保健品领域,为今后的分离纯化及活性测定提供了实验原料。     

低聚壳聚糖的复合酶法制备研究

通过对黏均分子量、酶活力、水解率和还原糖浓度的测定,研究了由商业α-淀粉酶、纤维素酶和果胶酶1∶1∶1(m/m/m)组成的复合酶降解壳聚糖的最佳工艺条件,结果表明:复合酶在酶底物比为1∶5(m/m)、pH 5.3、温度56℃的条件下,酶解2 h可得到分子量为1 000～4 000的低聚壳聚糖,且通过傅里叶红外光谱分析酶解后的产物结构无明显变化。     

蓝圆鲹蛋白的酶解过程动力学研究

为了探索定向制备蓝圆鲹蛋白活性多肽的工艺条件,进行了酶解体系的动力学研究。蓝圆鲹蛋白经碱性蛋白酶在一定条件下作用可得到活性多肽。采用pH-stat法建立了蓝圆鲹蛋白-碱性蛋白酶酶解体系的酶解反应动力学模型。利用高效体积排阻色谱分析了不同水解度下酶解产物中多肽片段族分子量的分布,并采用3-D图形表达了酶解过程中水解度-多肽片段族分子量-质量百分数之间的变化关系,经Matlab数学拟合得到了酶解过程中多肽片段族组分百分数与水解度和分子量的关系。验证实验表明,建立的3-D动力学模型与实际水解过程基本吻合,该模型揭示了酶解过程中多肽分子量组成的变化规律,可对酶解体系进行实时定量分析和动态表征,此酶解过程动力学研究也有利于定量获得定向目标产物。     

Purification and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk

The protein extracted from lecithin-free egg yolk, normally discarded by lecithin processing plants, was hydrolyzed with the aid of Alcalase, a commercial enzyme. The hydrolysate was separated through a series of ultrafiltration membranes with molecular weight cutoffs of 10, 5, and 1 kDa; and three types of permeates including 10 K (permeate from 10 kDa), 5 K (permeate from 5 kDa), and 1 K (permeate from 1 kDa) were obtained. The antioxidative efficacy of hydrolysates so obtained was investigated and compared with α-tocopherol. Furthermore, two different peptides showing strong antioxidative activity were isolated from the hydrolysates by using consecutive chromatographic methods including ion exchange chromatography on a SP-Sephadex C-25 column, gel filtration on a Sephadex G-25 column, and high-performance liquid chromatography on an octadecylsilane column. The purity of the peptides was identified using capillary electrophoresis. The isolated peptides were composed of 10 and 15 amino acid residues, and both contained a leucine residue at their N-terminal positions.     

Application of ultrafiltration to the preparation of defined hydrolysates of bovine haemoglobin

Many uses of protein hydrolysates have been developed and applied to areas such as nutritional therapy, culture media, and the isolation of biologically active peptides. All these applications need carefully controlled and characterized hydrolysates. In order to produce such a type of hydrolysate, it is possible to use haemoglobin which is a very well defined and constant protein source. Enzymic hydrolysis of haemoglobin by pepsin was carried out at pilot-plant scale in an ultrafiltration reactor with mineral membranes. The object was to obtain a reproducible, decolorized, salt-free enzymic hydrolysate. Two types of membranes were tested having 10000 dalton (M5 type) and 20000 dalton (M4 type) cut-offs. Little significant difference was observed in the final products when both types of membranes were used. Reproducibility of hydrolysates was verified by amino acid analysis and gel filtration chromatography. The haemoglobin hydrolysates produced contained more than 90% protein and are especially suitable for fine applications.     

Analyzing molecular weight distribution of whey protein hydrolysates

Process parameters on enzymatic hydrolysis and molecular weight (MW) distribution of whey protein hydrolysates were investigated. Whey protein hydrolysates were first gained by the alkaline protease alcalase for 7 h at temperature (50 °C), pH (8.0) and E/S (3%). The diversification of the hydrolysis degree and dissociative amino acid content was investigated during the whey hydrolysis. The dissociative amino acid content was 56.09 μmol/mL with the hydrolysis degree of 20.04%. The results of Sephadex G25 washing and high performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS) indicated the molecular weight distribution of whey protein hydrolysates ranged from 300 to 1400 Da, and most of whey peptide was under 1000 Da.     

Product distribution of casein tryptic hydrolysis based on HPSEC analysis and molecular mechanism

Tryptic hydrolysis of whole casein was analyzed by high performance size exclusion chromatography (HPSEC) in combination with the degree of hydrolysis (DH). In terms of chromatograms obtained at different DH values and mass percentage calculated equations established by the normalization method, the complex process of enzymatic reaction and the molecular mass distribution of multiple hydrolysates were quantitatively characterized via 2-D plot. Based on the information of casein micelle structure, the possible reaction mechanism was deduced from a series of chromatographic results and experimental analysis. Taking into account the primary structure of whole casein and the target amino acid of trypsin, the distribution of theoretical peptides was accurately calculated by determining the split sites of complete enzymatic hydrolysis. According to the relationship between retention time and molecular mass, the corresponding chromatogram absorption peaks of active peptides in hydrolysates were identified, and caseinophosphopeptides (CPPs) sequence was also characterized.     

Films made from polyethylene‐co‐acrylic acid and soluble biopolymers sourced from agricultural and municipal biowaste

Blends were obtained from polyethylene‐co‐acrylic acid (PEAA) with 248 kDa molecular weight and two water soluble biopolymers isolated from the hydrolysate of postharvest tomato plant and urban biowaste compost. The two hydrolysates were constituted respectively from a polysaccharide (SP) with 27 kDa molecular weight and a lignin‐like polymer (LP) with 75 kDa molecular weight containing aliphatic and aromatic C moieties substituted by carboxyl, hydroxyl, and amino groups. Evidence was obtained for reactions occurring between the biopolymers and the synthetic polymer leading to new polymers with 151 to 1243 kDa molecular weights. The thermal and mechanical properties of the blends were studied. Compared with neat PEAA, the PEAA‐LP blends containing 5 to 10% LP exhibited 2 to 5× higher molecular weights, 10 to 50% lower crystallinity, 2 to 6× higher Young's modulus, over 3× higher stress at yield point and somewhat lower strain at break (55–280% vs. over 300%). On the contrary the PEAA‐SP blends exhibited 6 to 13% lower crystallinity and the same mechanical properties as neat PEAA. The results offer scope for investigating biopolymers sourced from other biowastes to understand more the reasons of the observed effects and exploit their full potential to modify or to replace synthetic polymers. Perspectives of economic and environmental benefits are discussed. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41909.     

Enzymatic activity of Campylobacter jejuni hippurate hydrolase

The hippurate hydrolase enzyme of Campylobacter jejuni was expressed in Escherichia coli as a six-histidine-tagged fusion protein. The purified recombinant enzyme was characterized to gain an understanding of the structure and activity of the hippurate hydrolase. The recombinant enzyme had a native molecular mass of 193+/- 11 kDa a reduced molecular mass of 42.4+/- 0.8 kDa, and possessed 1.98+/- 0.68 molecules of zinc per enzyme subunit molecule, suggesting that it was a homotetramer with two associated zinc ions. The enzyme was a metallocarboxypeptidase that was sensitive to silver, copper and ferrous ions, and displayed optimal activity at pH 7.5 and 50 degrees C. It hydrolyzed carboxypeptidase substrates in vitro, displaying its highest activity against N-benzoyl-linked small aliphatic amino acids. A high proportion of the enzyme structure consisted of highly ordered alpha-helix and beta-sheet sequences. An alignment of the amino acid sequence of the hippurate hydrolase enzyme with those of related enzymes with similar activities revealed several conserved amino acids, which might be involved in enzyme catalysis or metal ion binding for the enzyme. Site-directed mutagenesis of the recombinant enzyme demonstrated that the Asp(76), Aps(104), Glu(134), Glu(135), His(161) and His(356) positions were important for the catalytic activity of the enzyme.     

Color control of Japanese soy sauce (shoyu) using membrane technology

The present study systemically decolorized soy sauce using a membrane process to analyze the separation mechanism. An ultrafiltration (UF) membrane (NTU-2120) exhibited only slight decolorization ability. A nanofiltration (NF) membrane with a lower molecular weight cut-off and produced by sulfonated polysulfone (NTR-7400 series) rather than polyvinyl alcohol/polyamide (NTR-7250) had higher decolorization ability. The NF membranes rejected total nitrogen by 17–24%, unsalted soluble solid content by 24–32%, reducing sugar by 25–43%, and amino acids by 10–25%. The NTR-7400 series membrane rejected lactic acid by 6–9%, and pyroglutamic acid by 11–21%; other quality indexes were maintained. In the NF membrane processes, higher rejection of acidic amino acids than neutral and base amino acids was observed. The separation performance was governed by the electrical effect as well as the sieve effect. Soy sauce color could be controlled by blending NF membrane-processed soy sauce with feed soy sauce. Color can be matched to preference in accordance with dishes by suitably blending NF membrane-processed soy sauce with feed soy sauce.     

Sphingomyelin Deacylase,the Enzyme Involved in the Pathogenesis of Atopic Dermatitis,Is Identical to the β-Subunit of Acid Ceramidase

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the β-subunit of aCDase, which consists of α- and β-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the β-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the β-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading β-subunit that evokes the ceramide deficiency in AD skin.     

Extraction of amino acids from protein hydrolysates by electrodialysis

Protein hydrolysates were obtained by acid hydrolysis from animal or human residues, such as poultry feathers, ox blood and human hair. After neutralization and discolouration with active charcoal, the hydrolysates were treated by successive electrodialysis (ED) in order to extract amino acids into several fractions. The current density and pH were optimized for each ED operation performed with preindustrial pilot scale equipment. The first step was the demineralization of amino acid mixtures using an ED stack with two compartments. The salt removal was achieved with extraction degrees higher than 90% and current efficiencies of about 80%. In the most favourable case, the amino acid losses did not exceed 10%. The second step was the extraction of the charged amino acids using an ED stack with four compartments. Three fractions were obtained, corresponding to the acidic, basic and neutral amino acids. The extraction degrees varied from 80% to 100%. In the third step, the fractionation of basic amino acids on the one hand, and neutral amino acids on the other hand, was carried out with enrichment degrees varying from 50% to 80%. © 1998 SCI     

Cloning and sequence analysis of D-erythrulose reductase from chicken: its close structural relation to tetrameric carbonyl reductases

Sequence analysis of a cDNA for D-erythrulose reductase fromchicken liver showed that the deduced open reading frame encodesthe protein with a molecular mass of 26 kDa consisting of 246amino acids. Although the reductase shares more than 60% identityin the amino acid sequence with the mouse tetrameric carbonylreductase, these two enzymes have many biochemical differences;their substrate specificity, subcellular localization, organdistribution, etc. A three-dimensional structure of D-erythrulosereductase was predicted by comparative modeling based on thestructure of the tetrameric carbonyl reductase (PDB entry =1CYD). Most of the residues at the active site (within 4 Åfrom the ligand) of the carbonyl reductase were also conservedin the D-erythrulose reductase. Nevertheless, Val190 and Leu146in the active site of the tetrameric carbonyl reductase weresubstituted in the D-erythrulose reductase by Asn192 and His148,respectively. The substitutions in the active sites may be relatedto the difference in substrate specificity of the two enzymes.The phylogenic analysis of D-erythrulose reductase and the otherrelated proteins suggests that the protein described as a carbonylreductase D-erythrulose reductase.