牙鲆（Paralichthys olivaceus）中不同亚型小清蛋白的提纯与鉴定

以牙鲆（Paralichthys olivaceus）为研究对象,以Tris、甘氨酸和二硫苏糖醇的混合溶液为提取液,加入不同量的硫酸铵分级纯化小清蛋白,应用免疫印迹法、酶联免疫法及质谱法进行鉴定。最终提纯得到3 种不同分子质量的蛋白（PVI、PVII和PVIII）,所得蛋白纯度大于90%,血清学实验证明其与小清蛋白特异性IgG及IgE抗体均有结合能力,质谱鉴定得到PVII和PVIII分子质量分别为12 151 Da和11 645 Da,pI值分别为5.14和4.69。该方法满足牙鲆小清蛋白提取纯化要求,得到的3 种蛋白是牙鲆小清蛋白的不同亚型,且均具有过敏原性。     

Purification and Characterization of Aminopeptidase from Chicken Intestine

ABSTRACT: An aminopeptidase was purified 839-fold with 15% recovery from chicken intestine by a procedure involving ion exchange, gel filtration, hydrophobic interaction and affinity chromatography. The enzyme is a heteridimer of subunits 94000 Da and 66000 Da. The substrate specificity of the enzyme was in the order ala > arg > leu-β-naphthylamide. The enzyme was strongly inhibited by bestatin, puromycin, and 1, 10-phenanthroline. The aminopeptidase showed maximum activity at pH 6 and 40 to 50 °C. The enzyme significantly differs from the hitherto known major classes of aminopeptidases from chicken tissues in terms of molecular weight and biochemical characteristics.     

微生态制剂对牙鲆幼鱼蛋白酶和生长的影响

试验采用正交实验的方法,确定了微生态制剂的使用方式、使用间隔时间、不同菌种的比例和使用浓度的最佳方案,以提高效率、降低成本。结果表明,微生态制剂能显著提高牙鲆幼鱼消化道的蛋白酶活性。同时在水中和饲料中使用微生态制剂,可以在短时间内大幅度提高牙鲆幼鱼消化道蛋白酶活性,最佳方案是投水兼投喂、间隔1周、1芽∶10乳、浓度为1×105个/m l;养殖水体换水频率较高时,可采用这种方案。对于换水频率较低的养殖水体,最佳方案是只在水中使用、间隔1周、10芽∶1乳、浓度为1×105个/m l。微生态制剂对牙鲆幼鱼的生长有显著的促进作用,试验组的平均体重比对照组高出26.86%。     

一种米曲霉耐盐蛋白酶的纯化及酶学性质分析

采用硫酸铵沉淀、Q-HP阴离子交换柱和Superdux 75凝胶柱等技术,从酱油大曲中纯化得到一种耐盐蛋白酶,经飞行时间质谱鉴定为钙蛋白酶RIM13,属于一种半胱氨酸蛋白酶。该蛋白酶的最适温度为50?℃;最适pH值为6.5;稳定温度为40?℃;稳定pH值为7.0;Mn2＋促进蛋白酶活力,Fe3＋、Fe2＋、Cu2＋、Ca2＋、K＋、Na＋抑制蛋白酶活力,以上金属离子对蛋白酶二级结构也产生不同程度的影响;米氏常数Km为2.43?g/L,最大反应速率Vm为103.09?mg/（L·min）。在5、10?g/100?mL和15?g/100?mL的NaCl质量浓度条件下,蛋白酶保留的酶活力分别为77.22%、54.39%以及41.15%。该蛋白酶在高盐度环境下保持较高的酶活力,因此具有潜在的工业应用价值。     

罗非鱼肠道蛋白酶的分离纯化及部分性质研究

目的:以罗非鱼加工下脚料肠道为原料,分离提取其中的蛋白酶,并对其部分性质进行研究,旨在提供一种简便的食品级蛋白酶制备方法.方法:用茶多酚络合沉淀,Sephadex G-50分离纯化蛋白酶,SDS-PAGE检测分离效果及相对分子质量;以酪蛋白溶液为底物,采用Folin-酚法测定蛋白酶活力.结果:经茶多酚络合沉淀,得到一种复合蛋白酶,命名为TP-肠道蛋白酶;经Sephadex G-50凝胶层析除去茶多酚,得到游离肠道蛋白酶.TP-肠道蛋白酶的最适温度50℃,pH7.2～9.6,米氏常数Km=1.125×103mg/L;游离肠道蛋白酶的最适温度50℃,pH7.2～10.0,米氏常数Km=8.5×102mg/L.结论:TP-肠道蛋白酶和游离肠道蛋白酶均为复合蛋白酶,有望开发成食品级蛋白酶制剂.     

表面活性剂稳定性碱性蛋白酶纯化及性质研究

从地衣芽孢杆菌(Bacillus licheniformis)XG12发酵液中分离纯化表面活性剂稳定性碱性蛋白酶,并对酶学性质进行研究。利用硫酸铵分级盐析、DEAE-Sepharose阴离子交换层析和Sephadex G-75分子筛凝胶过滤层析等方法,分离纯化到了均一的酶蛋白,酶纯度提高了42.6倍,回收率为25.3%。SDS-PAGE及Sephadex G-75分子筛凝胶过滤层析显示酶蛋白为单亚基蛋白,分子质量约为29.5kD。在pH7.0～11.0范围内酶活性及稳定性较高,最适作用pH值为10.0,最适作用温度40℃。Mg2+、Ca2+及Mn2+对酶有明显激活作用。丝氨酸蛋白酶特异性抑制剂强烈抑制酶活性,表明所纯化到的蛋白酶为丝氨酸蛋白酶。酶分别对终质量浓度为0.1g/100mL的阴离子表面活性剂SDS、阳离子表面活性剂CTAB和体积分数为1%的非离子型表面活性剂Tween-80、Tween-20、Triton X-100以及氧化剂均具有很强的稳定性。     

Partial Purification and Characterization of a Neutral Protease from Bovine Skeletal Muscle

A method has been developed for the partial purification of a calcium activated neutral protease from bovine muscle, since application of methods previously used for rabbit or porcine muscle gave little or no yield. The new method involves extraction with phosphate-buffered KCl, saling out with (NH₄)₂SO₄, affinity chromatography on mercurial agarose followed by gel filtration. The bovine protease required calcium ion and reducing agent for activity with a pH optimum at 7.5 and hydrolyzed myofibrillar proteins.     

Preparation and Characterization of an Olive Flounder (Paralichthys olivaceus) Skin Gelatin and Polylactic Acid Bilayer Film

Olive flounder skin gelatin (OSG) was used as a film base material. A bilayer film of OSG and polylactic acid (PLA) was prepared using solvent casting method to enhance the film properties. Physical properties of the OSG–PLA film were increased compared with the nonaugmented OSG film. In particular, the PLA lamination decreased water vapor permeability from 2.17 to 0.92 × 10^?9 g·m/m²·s·Pa, as well as of the water solubility from 16.62% to 9.27%, in the bilayer film relative to the OSG film. The oxygen permeability of the OSG–PLA bilayer film was held low by the OSG film, compensating for the high oxygen permeability of the PLA layer. Therefore, the OSG–PLA bilayer film with its enhanced physical properties and high water and oxygen barrier properties can be applied as a food packaging material.     

Stabilization and Partial Purification of a Protease from Ginger Rhizome (Zingiber offinale Roscoe)

ABSTRACT: Ginger protease (GP) or zingibain is of interest as a meat tenderizing agent. The objective of this research was to investigate food-compatible methods for stabilizing GP during storage or enzyme fractionation. Crude GP extracted from fresh ginger had a half-life (t_1/2) of 2.1 (±0.16) d at 5°C decreasing to 20 min at 30°C. Addition of ascorbate (0.2% w/v) increased the t_1/2 for GP from 2 to 20 d at 5°C. Dithiothreitol or Ethylenediaminetetraacetic acid (EDTA) had no effect on GP stability. Acetone powder preparations from ginger yielded GP with t_1/2 of 18 mo at 5°C. Crude GP extracted from acetone powder was sufficiently stabilized to allow fractionation by ion exchange chromatography without the addition of toxic or expensive additives. GP was partially purified 252-fold with a recovery of 61%. The nomimal molecular weight of GP was 34.8 kDa compared with 25.1 kDa for papain. This work shows that the stability of GP can be greatly improved, increasing its attractiveness as a commercial product. Some possible routes of GP deactivation and stabilization are discussed.     

Enterococcus faecalis RQ15产低温中性蛋白酶的纯化及酶学性质

采用DEAE Sepharose Fast Flow和SuperdexTM 75对Enterococcus faecalis RQ15产蛋白酶进行纯化和酶学性质研究。SDS-PAGE测定该蛋白酶分子质量为32.4kD,最适作用温度35～40℃,最适pH7.5。20～40℃之间酶活较高,pH值耐受范围广泛,具有低温蛋白酶的特征。Zn2+对蛋白酶有激活作用,Ag+、Hg2+和EDTA-Na2对酶有显著抑制作用。纯酶最适作用条件下对酪蛋白底物的Km和Vmax分别为1.31×10-4mol/L和6.92×10-6mol/(L ·s)。     

一株深海来源苏云金芽孢杆菌SWJS07所产蛋白酶的分离纯化及性质研究

利用超滤、硫酸铵盐析、DEAE Sepharose Fast Flow阴离子交换柱层析、Sephadex G-75分子筛柱层析对一株南海深海来源菌苏云金芽孢杆菌(B.thuringiensis)SWJS07所产蛋白酶进行分离纯化,纯化后经SDS-PAGE鉴定达到电泳纯,相对分子质量为37.0 k Da,酶的比活力提高了6.39倍,回收率为37.14%。研究其酶学性质表明,该蛋白酶最适催化温度为55℃,在30℃~45℃下稳定性较高,保温300 min残留酶活在80%以上;最适p H 6.5,在p H 6.0~9.0蛋白酶稳定,4℃放置24 h残留酶活在80%以上;2 m M Ca2+、Mn2+对蛋白酶有不同程度的激活作用,而Hg2+、Cd2+、Al3+则强烈地抑制蛋白酶活;当在蛋白酶中添加2 m M Ca2+、Mn2+时,其最适催化温度分别为60℃和55℃,蛋白酶活分别提高了32.86%和28.35%,60℃保温30 min相对酶活基本保持不变,与纯酶(相对酶活残留21.02%)相比蛋白酶的热稳定性显著提高;EDTA-Na2可强烈抑制蛋白酶活,推测该蛋白酶属于金属蛋白酶。     

Identification and Partial Characterization of a Bacteriocin‐Like Inhibitory Substance (BLIS) from Lb. Bulgaricus K41 Isolated from Indigenous Yogurts

Forty‐two strains of Lactobacillus bulgaricus isolated from locally made yogurts were examined and compared for bacteriocin producing ability using spot on lawn assay which improved by taking photo and image processing. Lb. bulgaricus K41 exhibited the highest inhibition level against indicators. K41 Bacteriocin‐like inhibitory substance is sensitive to proteolytic enzymes (proteinase K, pepsin, and trypsin) but α‐amylase makes slight reduction in its activity and it is resistant to lipase. This antibacterial peptide is extremely heat‐stable (121 °C for 15 min) and remains active over a wide pH range (pH = 2 to 10); also nonionic detergents (Tween‐20, Tween‐80, and Triton X100) showed no effect on its activity. The inhibitory spectrum is against Gram‐positive bacteria (except Staphylococcus aureus) with extremely antilisterial activity and it is almost ineffective against Gram‐negative bacteria. The mode of its action was identified as bactericidal against Listeria monocytogenes. The properties of K41 bacteriocin‐like inhibitory substance add to its safety as a biopreservative produced by a generally recognized as safe (GRAS) bacterium suggesting it can be used in hurdle technology for ready‐to‐eat foods as one of the main sources of Listeria contaminations.     

章鱼消化道蛋白酶的分离纯化及性质

以章鱼加工下脚料消化道为原料,经硫酸铵沉淀、纤维素CM-52阳离子交换层析、DEAE-Sephadex A50阴离子交换层析、SDS-PAGE电泳等方法,从中提取纯化出一种蛋白酶电泳纯样品OP-I,并对其性质进行研究。结果表明：该酶分子质量为80.5kD。最适反应温度为55～60℃,pH值为7～9。金属蛋白酶抑制剂(EDTA)可以完全抑制该酶的活性。Mn2+、Ca2+、Mg2+对OP-I有激活作用,酶促动力学研究显示其米氏常数Km为0.33mmol/L,Vmax为66.7mg/(mL ·min)。     

基因重组牙鲆生长激素(r-fGH)对牙鲆幼苗生长的促进作用

将含基因重组牙鲆生长激素(r-fCH)的毕赤酵母直接作为饵料源,研究r-fGH促进牙鲆幼苗生长的水平依存关系.结果表明,6周喂养后,含r-fGH的酵母组,无论是低水平还是高水平,均对牙鲆幼苗的生长有显著的促进作用,鱼体的相对体重增长率、相对体长增长率、食物转化率和肥满度均增加显著.     

Protein degradation of olive flounder (Paralichthys olivaceus) muscle after postmortem superchilled and refrigerated storage

The aim of this study was to investigate the effect of superchilling at ?2°C in comparison with refrigerated storage at 4°C on the protein degradation of olive flounder (Paralichthys olivaceus) muscle. Flounder muscle softened and shear force value decreased markedly (P < 0.05) with prolonged storage time, while values of electrical conductivity, TCA-soluble peptide, free amino acids, and proteolysis index increased (P < 0.05). The changes were slowed down significantly in samples superchilled at ?2°C (P < 0.05). The fracture of muscle fibre and formation of cracks were accelerated in the samples refrigerated at 4°C, and intercellular spaces were observed after 9 days of storage. Moreover, protein bands of myosin heavy chain (MHC), actin, tropomyosin, 97 kDa, 50 ~ 60 kDa and 35 ~ 36 kDa occurred in varying degrees of degradation with storage time. The results demonstrated that significant postmortem degradation of muscle proteins occurred with extending storage time, while the changes were retarded obviously in samples during superchilled storage.