Immobilization and characterization of ficin

Fig tree latex (ficin) was immobilized on Celite by adsorption. The free and immobilized ficin were utilized in the production of teleme (a Turkish milk product). After immobilization, the optimal temperature of ficin was shifted from 60 to 80 degrees C. Both free and immobilized ficin exhibited higher milk clotting activity in the acidic pH range. Increase in solid content of milk affected milk clotting activity in opposite direction. When the amount of either free or immobilized enzymes doubled, clotting timereduced nearly 1.6 and 1.7 fold for free and immobilized enzyme, respectively. Use of immobilized enzyme in teleme production gave better results in terms of chemical and sensory properties compared with teleme made by free enzyme. Syneresis in teleme reduced from 70% to 60% depending on immobilization. Both acidity and bitter taste of teleme produced from free ficin was due to extensive protein hydrolysis by proteolytic fractions.     

Purification of Amylase from Honey

The major amylase in honey was concentrated by ultrafiltration, isolated by ultracentrifugation and gel filtration, and purified by ion‐exchange chromatography. The amylase activity was in the flow‐through fraction of the anion‐exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzyme fraction from the anion‐exchange chromatography was loaded onto a cation‐exchange column, and the amylase activity was eluted as a single band at 50 mM NaCl. The purification factor after this step was 531‐fold. The purified enzyme was an a‐amylase, as determined by thin‐layer chromatography, with a molecular weight of 57000 Da according to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The results supported the concept that amylase in honey had a high degree of similarity with bee amylase.     

Inhibition of endive (Cichorium endivia L.) polyphenoloxidase by a Carica papaya latex preparation

When endive polyphenoloxidase (PPO) was incubated with a crude papaya latex extract, it rapidly lost its activity. Inactivation was ascribed to thermostable nonenzymatic factors of low molecular weight. These factors were partially purified by a two step protocol including gel filtration chromatography on Biogel P2 and ion exchange chromatography using DEAE Sephadex A25. The PPO-inactivation rate was first order, when either inactivating agent or proton concentration was evaluated. Inactivation could be partially reversed by CuSO₄, which suggested that the inactivating factor(s) bound to the copper site of the enzyme. On a more rapid time scale than inactivation, papaya latex extract acted also as a weak noncompetitive PPO inhibitor.     

Expression and purification of the recombinant mustard trypsin inhibitor 2 (MTI2) in Escherichia coli

The mustard trypsin inhibitor 2, MTI2, was expressed in Escherichia coli. A specific procedure for its production and purification is described. The recombinant protein was recovered by protein extraction from the insoluble fraction, then renatured and purified by ion exchange and gel filtration chromatography. Finally, the inhibitory activity against trypsin was also determined.     

榆干离褶伞菌丝体中溶栓酶的分离纯化

采用离子交换层析、凝胶过滤层析及快速蛋白液相色谱等蛋白质分离纯化技术,从榆干离褶伞菌丝体中分离纯化了一种溶栓酶。实验结果表明,其分子量约为50ku,比活性为750.6Unit/mg,活性得率为12.5%。榆干离褶伞很有潜力成为治疗血栓的新的药物来源。     

热带假丝酵母木糖醇脱氢酶的分离纯化

探讨了热带假丝酵母中木糖醇脱氢酶的分离纯化工艺。分步采用硫酸铵分级沉淀、DEAE-Sepharose离子交换层析、Sephacryl S-200凝胶层析以及Hi Trap Blue HP亲和层析等纯化方法,得到电泳纯的木糖醇脱氢酶。经SDS-PAGE和native-PAGE分析表明,木糖醇脱氢酶分子质量约为90ku,由2个分子质量为45ku的亚基组成。最终酶蛋白纯化倍数达到60·8倍,回收率为10·9%。     

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。     

Winged bean lipoxygenase—Part 1: Isolation and purification

Lipoxygenase was isolated and purified from dried winged bean seeds by ammonium sulphate fractionation, gel filtration, DEAE-Sephadex ion exchange chromatography and hydroxyapatite chromatography. Two major isoenzymes, FI and FII, were separated by ion exchange chromatography and were further purified by elution through a hydroxyapatite column. These resulted in a 105- and 171-fold purification and 7% and 9% recovery for FI and FII, respectively. FI and FII had similar Rf values of 0.25 on polyacrylamide gel electrophoresis. A minor band of Rf 0.01 was detected in ammonium sulphate fractions but was not further enriched and purified in succeeding steps.     

蛋清抗氧化肽分离纯化及结构鉴定

为了获得高活性、高纯度的蛋清抗氧化肽,以蛋清酶解物为原料,依次釆用超滤、离子交换色谱、凝胶色谱分离纯化抗氧化活性较强的肽段,运用基质辅助激光解吸离子化质谱解析肽链的氨基酸序列。结果表明:超滤法分离纯化EWPH所得的三个组分中,EWPH-Ⅲ(M_W<3 kDa)组分的DPPH自由基清除率最高,达到79.62%。离子交换层析分离纯化EWPH-III所得到的碱性组分B的DPPH自由基清除率最高,达到82.05%。凝胶过滤色谱分离EWPH-III-B所得到4组分中E组分的DPPH自由基清除率最高,为88.49%。高活性高纯度EWPH-III-B-E组分的相对分子质量为237.575,该二肽的氨基酸序列为丙氨酸-甲硫氨酸。     

甘薯叶过氧化物酶的分离纯化及其部分性质研究

经硫酸铵分级沉淀、DEAE-Sepharose离子交换层析、Sephacryl S-200凝胶过滤层析,从甘薯叶中得到过氧化物酶(POD)电泳纯制品。该酶比活力为91923.14U/mg,纯化倍数为255.69,回收率为1.59%。该酶分子量约为35kD,最适pH值为5.6,最适温度为60℃。该酶在20～50℃、pH4～8内稳定。以不同浓度H2O2为底物在pH7.2和25℃下测得该酶Km值为0.291mol/L。低浓度草酸、尿素、Li+、Na+、K+、Mg2+等对该酶有激活作用;SDS、KSCN、抗坏血酸(AsA)、Mn2+等对该酶有抑制作用;甲醇、乙醇、乙二醇、异丙醇对POD均有一定抑制作用,其抑制作用强弱顺序为异丙醇>乙醇>甲醇>乙二醇。实验表明,该酶稳定性较强。     

祁连山荷叶离褶伞菌丝体LML的分离纯化及其凝血活性检测

以荷叶离褶伞菌丝体为材料,利用DEAE-Sepharose F.F.色谱和SephadexG-100色谱技术,分离获得新的LMLLML。根据凝胶过滤色谱和SDS-PAGE电泳结果,确定LML是中性糖含量为10.36%、分子量约为48.2ku单亚基糖蛋白。凝血活性检测结果表明,LML对兔血红细胞具有凝聚活性,这种活性在LML浓度为500μg/mL、温度为20⁷0℃、pH3¹0的条件下稳定;其凝血活性可被葡萄糖（最小浓度为25mmol/L）和麦芽糖（最小浓度为12.5mmol/L）所抑制;当LML浓度≥250μg/mL时,其凝血活性不受Ca2+、Mg2+和Zn2+的影响。      

Antioxidant Activity and Amino Acid Profiling of Protein Hydrolysates from the Skin of Sphyraena barracuda and Lepturacanthus savala

The antioxidant activity of protein hydrolysates prepared from the skin of Sphyraena barracuda (Seela fish) and Lepturacanthus savala (Ribbon fish), using the commercial enzymes pepsin, trypsin, and papain were determined. The protein hydrolysate showed high antioxidant activity in which trypsin hydrolysate of the skin of both seela and ribbon fish proved good DPPH scavenging activity with 66 and 60% (p < 0.05), respectively. These active hydrolysates were purified using fast protein liquid chromatography on ion exchange and gel filtration chromatography procedure and the active purified fractions were determined using electron spin resonance spectrophotometer against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. Further the purified fractions were analyzed for amino acid composition using high performance liquid chromatography and it consisted of antioxidant inducing amino acids along with a considerable quantity of essential amino acids.     

内生多黏类芽孢杆菌纤溶酶的纯化及其体外溶栓作用

植物内生菌株 EJS-3 发酵液经离心除菌,硫酸铵分级沉淀,Hiprep phenyl FF 疏水层析,RESOURCEM Q离子交换层析和Sephacryl S-300HR 凝胶过滤,获得电泳纯的多黏芽孢杆菌纤溶酶(PPFE-I),聚丙烯酰胺凝胶电泳(SDS-PAGE)测定其分子质量为63kD,高效液相色谱法(HPLC)鉴定其纯度为94.1%。每升发酵液中可获得1.6mg 活性蛋白,每毫克蛋白活力达2096IU,纯度提高了14.5 倍,回收率为3.3%。纯化后的纤溶酶PPFE-I 具有明显的体外溶栓作用。     

罗汉果蛋白酶的分离纯化和部分性质研究

对罗汉果蛋白酶的分离纯化和部分性质进行研究.利用硫酸铵盐析、离子交换和凝胶过滤柱层析对罗汉果蛋白酶进行了分离纯化,对纯化所得的酶进行了分子量和等电点测定.结果表明,纯化酶为电泳纯,回收率为4.2％,纯化倍数为31.1,凝胶过滤层析和SDS-PAGE电泳测得酶的分子量分别为40.3 ku和39.0 ku,等电聚焦电泳测得其等电点为8.55.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

ISOLATION AND ANALYSIS OF A NOVEL ACIDIC POLYSACCHARIDE WITH GLUCOKINASE-STIMULATING ACTIVITY FROM COARSE GREEN TEA

Water‐soluble polysaccharides from coarse green tea were separated by anion‐exchange chromatography into five fractions (fraction A [FA], fraction B, fraction C [FC], fraction D and fraction E). Two of these fractions, FA and FC, contained significant glucokinase‐stimulating activity (P < 0.05). The major component, FC, showed the most activity, and thus, was further purified by gel filtration chromatography, thereby obtaining fraction C‐1 (FC‐1) and fraction C‐2 (FC‐2). The biological activity of the two fractions was investigated, and FC‐1 displayed higher glucokinase‐stimulating activity (P < 0.01). Chemical tests combined with IR and UV spectroscopy revealed that FC‐1 is an acidic polysaccharide without conjugation to protein. Sugars of FC‐1 are composed of rhamnose, arabinose, mannose, glucose and galactose in the ratio of 12.57:22.95:4.4:39.34:20.77. Uronic acid analysis by ion chromatography showed that FC‐1 contains 8% galacturonic acid, and its molecular weight was estimated to be approximately 6 × 10⁴ using a Sephacryl S200 column. These results are different from the observations previously reported, therefore suggesting that FC‐1 is a novel polysaccharide.     

Nomilin Acetyl-Lyase, a Bacterial Enzyme for Nomilin Debittering of Citrus Juices

Nomilin acetyl-lyase, an enzyme responsible for the conversion of bitter nomilin to nonbitter obacunone was purified from cell-free extracts of Corynebacterium fuscians by (NH₄)₂SO₄ precipitation, followed by HPLC gel filtration and ion exchange chromatography. The electrophoretically pure preparation represented a 1050-fold increase in specific activity with a recovery of 38%. The enzyme, (MW 90,000) required sulfhydryl groups for its catalysis, had a pH optimum of 8.5, a K_m of 38.1 PM, and a V_max of 4.83 μmoles/min. Obacunone A-ring lactone hydrolase, which catalyzes the conversion of obacunone to obacunoate, was also purified from the extract by HPLC gel filtration.     

耐酸性木聚糖酶在清酒酿造中的作用

从华根霉 (RhizopuschinensisY92 )发酵液中通过离子交换色谱和凝胶过滤色谱分离纯化获得一种耐酸性木聚糖酶R ,将它和另外 2种耐酸性木聚糖酶 :米曲霉 (AspergillusoryzaeRIB12 8)木聚糖酶B和白曲菌 (AspergilluskawachiiIFO43 0 8)木聚糖酶C分别应用于清酒酿造中 ,结果表明 ,木聚糖酶B可以促进米细胞的溶解 ,对于原料米的利用率有明显的提高 ,而木聚糖酶C和木聚糖酶R的作用则不太明显     

烟草内切多聚半乳糖醛酸酶抑制蛋白分离纯化研究

首次报道烤烟中存在ＰＧＩＰ，并以烤烟苗为材料通过硫酸铵沉淀、离子交换层析和分子筛凝胶过滤等步骤分离得到了ＰＧＩＰ，经高ｐＨＤｉｓｃ－ＰＡＧＥ和ＳＤＳ－Ｄｉｓｃ－ＰＡＧＥ分析为电泳纯，紫外吸收为典型的蛋白质吸收光谱。烤烟ＰＧＩＰ是一种糖蛋白，含糖量为６．０３％，等电点为６．４和６．１。     

莲子多酚氧化酶的分离与纯化

由多酚氧化酶（polyphenol oxidase,PPO）导致的酶促褐变严重影响莲子的外观。本研究旨在探讨莲子PPO的提取纯化方法,为研究其蛋白结构、进一步探究莲子褐变机理提供理论依据。以酶活力和酶比活力为指标,对比了提取PPO的四种方法,发现匀浆吸附后丙酮沉淀法提取莲子PPO可获得较高的酶比活力,达289.04 U/mg;采用单因素实验对该提取方法进行优化,得最佳提取参数为:料液比1∶5、提取时间2 h、p H=7、含2%PVPP和0.8%Triton X-100提取溶液,优化后PPO粗酶液酶比活力为294.40 U/mg;采用DEAE Sepharose Fast FLow阴离子交换层析、Sephadex G-75凝胶柱层析对粗酶液进行纯化,得到分子量大约为58 ku莲子PPO,酶比活力为7926.68 U/mg,纯化倍数为26.92,回收率为38.48%。