Purification and biochemical characterization of chymotrypsin from the viscera of Monterey sardine (Sardinops sagax caeruleus)

Chymotrypsin was isolated from the viscera of Monterey sardine by ammonium sulphate fractionation, gel filtration, and ionic exchange chromatography. The approximate molecular weight was 26,000 and its isoelectric point was about 5. Identity as chymotrypsin was established by its catalytic specificity for amide or ester bonds on the synthetic substrates succinyl-l-ala-ala-pro-l-pheilalanine-p-nitroanilide and benzoyl-l-tyrosine-ethyl-ester, showing esterase activity 3.2-fold higher than amidase. It was inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, partly inhibited by the specific chymotrypsin inhibitor N-toluenesulfonyl-l-phenylalanine chloromethyl-ketone, but not inhibited by EDTA or Benzamidine. Chymotrypsin showed its maximum activity at pH 8.0 and 50 °C for the hydrolysis of SAAPNA. The Michaelis–Menten constant was 0.074 mM with a catalysis constant of 18.6 seg⁻¹, and catalytic efficiency of 252 seg⁻¹ mM⁻¹. Results indicated that Monterey sardine chymotrypsin is a good catalyst and could be used as a biotechnological tool in food processing and using sardine industry wastes as a material for production of fine reagents.     

Biochemical characterization of an isoform of chymotrypsin from the viscera of Monterey sardine (Sardinops sagax caerulea), and comparison with bovine chymotrypsin

Chymotrypsin II from the viscera of Monterey sardine was characterized as an isoform of chymotrypsin I previously characterized from the same source and compared with bovine chymotrypsin. Chymotrypsin II had a molecular weight of 25,500 Da, similar to bovine chymotrypsin. The isoform identity as chymotrypsin was established by its catalytic specificity on the specific substrates succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and benzoyl-l-tyrosine ethyl ester, showing higher specific activity than bovine chymotrypsin. Both enzymes showed maximal activity at pH 8.0, chymotrypsin II being stable at alkaline pH while bovine chymotrypsin was stable at acid and alkaline pH. Optimum temperature was 45 °C for chymotrypsin II and 55 °C for bovine chymotrypsin. Both enzymes were inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, and partially by N-toluenesulfonyl-l-phenylalanine chloromethyl-ketone. This is valid only in specific conditions of this work. K_m and k_cat for chymotrypsin II were 0.048 mM and 4.8 s⁻¹, and 0.09 mM and 1.9 s⁻¹ for bovine chymotrypsin. Catalytic efficiency of chymotrypsin II was 4.8-fold higher than bovine chymotrypsin.     

Thermodynamic activation and structural analysis of trypsin I from Monterey sardine (Sardinops sagax caerulea)

In this work, we report the molecular characterisation of trypsin I (Try I) from Monterey sardine (Sardinops sagax caerulea). Aspects such as thermodynamic activation parameters, molecular model and cDNA-deduced amino acid sequence allow a more in depth understanding of its activity at low temperatures. The analysis of the thermodynamic activation parameters suggests that this molecule is a cold-adapted protease. From the molecular cloning, we deduced the amino acid sequence and predicted a theoretical structural model of sardine Try I with a classical trypsin fold. Cold-adaptation of this enzyme probably comes from amino acid replacement of key residues to improve flexibility at low temperature, thus increasing k_cat. The cold-adaptation of sardine Try I opens a wide range of biotechnological applications for this protease and also it is interesting from the structure function relationship point of view of serine protease proteins.     

两种脱毛蛋白酶活力的研究

本文对两种常用的脱毛蛋白酶（2709碱性蛋白酶与A．S1398蛋白酶）在不同pH值、温度等条件下的活力变化情况进行了研究。结果表明：2709蛋白酶的活力最适pH值为9．5～10，A．S1398蛋白酶为7．5～8．0且2709酶的pH值适应范围更广，对pH值的敏感度更低；两种酶活力的温度变化曲线具有相似性，温度越高，它们的活力衰减得也越快；两种蛋白酶受Mn抖强激活，Cu^2＋、Cr^3＋、Fe^2＋、Zn^2＋等离子则对其有抑制作用；表面活性剂如洗衣粉、平平加和JFC等对两种酶具有一定的激活作用。     

Extraction and fractionation of lipolytic enzyme from viscera of Monterey sardine (Sardinops sagax caerulea)

In this study, lipolytic activity of a semi-purified lipolytic enzyme (SLE) from the viscera of sardine ( Sardinops sagax caerulea ) was screened on the lipolysis of olive, Menhaden and sardine oil. A lipolytic enzyme was partially purified from the crude extract of sardine viscera by fractional precipitation followed by ultrafiltration (30 kDa). The main tissues found in sardine viscera were pyloric caeca (19.0% w/w), digestive tract (13.0% w/w), liver (4.8% w/w) and pancreas (1.5% w/w). Results show that pancreas had the highest lipolytic activity. There were no significant differences in lipolytic activity between pyloric caeca, intestine and liver ( P  < 0.05). Specific activity of the SLE increased 47.0-fold after extraction and fractionation, with a yield of 0.34% calculated for the whole viscera weight. Lipolytic activity of SLE from sardine viscera increased threefold when sardine oil was used as substrate. The results of this study confirm the potential importance of lipases from marine sources.     

罗非鱼骨骼肌中胶原蛋白分解酶的提取纯化及活性

新鲜罗非鱼肉匀浆离心后经硫酸铵分级沉淀、SephadexG-100凝胶柱层析、DEAESephadexA-50阴离子交换后,得到经SDS-PAGE证实的纯品.650g罗非鱼骨骼肌可制备总活力14835U,比活力526.4U/mg的胶原蛋白分解酶,纯化倍数为111.53倍.     

我国豆酱血管紧张素转换酶抑制活性的比较

文章对我国三种市售豆酱的血管紧张素转换酶(ACE)抑制活性及其主要化学组成进行比较分析,所试样品的IC50值分别为1.373、0.876和1.691 mg/mL,活性最高样品中的多肽含量为24.09g/100g dry matter,不同样品间多肽分布无明显差异。样品的ACE抑制活性应为多种活性物质综合作用的结果,对其中ACE抑制剂构效关系的研究可为我国豆酱功能性的研发提供指导。     

微生物发酵生产纤溶酶及其酶学性质的研究进展

本文介绍了微生物来源的纤溶酶发酵、酶活测定方法以及酶学性质方面的研究进展。     

米曲制备过程中酶活性的变化及制备工艺研究初报

以东北大米为原料、以清酒用种曲为菌株，研究了东北大米米曲制备过程中酶活性的变化及制备工艺。分别使用丸福种曲和沙加办吟酿用种曲进行制曲，分析了东北大米米曲的制备过程中的酶活力变化，确定了出曲时间，完善了生产工艺。使用3种商品种曲进行制曲，其中曲的品质测定结果为：接种丸福种曲，制成的米曲的糖化酶活力为404u／g，α-淀粉酶的活力为324u／g，酸性蛋白酶活力为385u／g；接种山田锦驯养种曲，制成米曲的糖化酶活力为414u／g，α-淀粉酶的活力325u／g，酸性蛋白酶活力为748u／g；接种ぴかみ吟酿用种曲，制成的米曲的糖化酶活力为409u／g，α-淀粉酶的活力为328u／g，酸性蛋白酶活力为376u／g。     

酶解法制备米糠膳食纤维

对米糠中膳食纤维的酶解法制备工艺进行了研究。通过单因素试验、正交试验和极差分析,得出了酶法制备米糠膳食纤维的最佳工艺参数。结果表明,在混合酶(α-淀粉酶与糖化酶质量之比为1∶2)用量0.4%、70℃处理45min,蛋白酶用量0.3%、50℃处理45min时,膳食纤维得率较高,膳食纤维中的酸性洗涤纤维含量达68.54%。因此,该酶法工艺为米糠膳食纤维的制备提供了依据。     

延迟冷却对钙激活酶m-calpain活性及牛肉嫩度的影响

本文通过对中国杂交黄牛（鲁西黄牛×西门塔尔）牛背最长肌中钙激活酶m-calpain活性及剪切力值的分析,研究了延迟冷却对牛背最长肌宰后成熟过程中m-calpain活性及牛肉嫩度的影响。结果表明,延迟冷却仅在宰后成熟1h,显著提高了m-calpain的活性（P〈0.05）;宰后成熟24h、3d时,与对照组之间差异不显著（P〉0.05）;试验结果证实,m-calpain在牛肉的嫩化过程中并不起主要作用,对牛肉的嫩度贡献不大。     

马铃薯块茎粗多酚体外抗氧化活性

对马铃薯块茎粗多酚抗氧化活性进行研究。采用乙醇、丙酮、蒸馏水3种溶剂提取马铃薯块茎中粗多酚类物质,通过粗多酚提取液对羟自由基(.OH)、超氧自由基(O2-.)、亚硝酸盐(NO2-)的清除和还原能力测定,比较3种酚提液的体外抗氧化活性,并测定其中总多酚含量。结果显示:3种粗多酚提取液总多酚含量为0.33～0.40 mg/mL;在不同抗氧化体系中,3种酚提液均具有良好的抗氧化活性,抗氧化能力的大小顺序为60%乙醇酚提液>60%丙酮酚提液>蒸馏水酚提液,抗氧化活性的大小与总多酚含量的高低呈正相关。     

干酪乳杆菌胞壁蛋白酶提取条件优化及其水解特性研究

干酪乳杆菌细胞壁蛋白酶是其蛋白水解系统中一个关键的蛋白酶.采用Ca2+-free法研究干酪乳杆菌细胞壁蛋白酶的提取方法,确定其最佳实验条件.菌体用含有30 mmol/L CaCl2的50 mmol/L Tris-HCl (pH 7.0)缓冲液洗涤3次,然后用含有50.03 mmol/LEDTA-Na2的50 mmol/LTris-HCl(pH6.98)缓冲液悬浮,39.75℃保温,60 min后取出离心(4 500 r/min、20 min、4℃),上清液即粗酶液.用粗酶液与纯化后的酶分别水解各种酪蛋白,粗酶较纯化后的酶能产生更高的ACE抑制能力,而纯化后的酶水解β-酪蛋白产生的抗ACE活性能力最强.     

固态发酵大豆生产豆豉纤溶酶的研究

以大豆为底物进行固态发酵生产纤溶酶，并对其固态发酵工艺进行了探讨。得出以下结论：最佳发酵时间28h，添加麦芽糖2％、NaCl 0.1％、Ca2^2 0.2mg／kg、Mg^2 0.3mg／kg最高酶活力可达到1256IU／g。豆豉提取物的最适存放温度为37℃以下，最适pH为8，Mn^2 、Ca^2 、Mg^2 对该酶活力有明显的激活作用，添加明胶对纤溶酶活力起稳定作用。PCMB、PMSF、胰蛋白酶对此酶活性抑制率几乎达100％，EDTA、β-巯基乙醇对此酶活性影响不大。     

茄子果实采后软化过程中细胞壁组分及其降解酶活性的变化

研究了细胞壁组分及其降解酶活性的变化与茄子果实采后软化的关系。结果表明,采后茄子果肉硬度随贮藏时间的延长而不断下降。贮藏期间果肉水溶性果胶(WSP)含量在贮藏前12天不断增加,之后快速下降,而共价结合型果胶(CSP)、半纤维素和纤维素等细胞壁组分含量持续减少。果肉果胶甲酯酶(PME)、多聚半乳糖醛酸酶(PG)和纤维素酶(CX)活性均呈先升高后下降趋势,分别在贮藏至第6、9、12天达到最大值;β-半乳糖苷酶(β-Gal)活性始终保持较高水平,且在整个贮藏期间活性变化不明显。相关性分析结果表明,CSP、半纤维素和纤维素的降解与采后茄子果实软化密切相关,PG和CX在茄子果实采后软化过程中起着重要的作用。     

Purification and Characterization of a Novel Protopectin-Solubilizing Enzyme from Aspergillus niger Z-25

A mutant, Aspergillus niger Z-25 derived from A. niger XZ-131 with N⁺ supplementation, produced a protopectin-solubilizing enzyme (protopectinase). The enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation and gel chromatographies on DEAE-Sephadex A-50 and Sephadex G-100 columns. The molecular weight of the protopectinase was estimated at 68.4 KD by SDS-polyacrylamide gel electrophoresis. The enzyme was stable from pH 3.0 to 7.0 and up to 60°C. The optimum pH and temperature for enzyme activity was 4.0 and 50°C, respectively. The purified enzyme had 268-U/mL PPase (protopectinase) activity on citrus protopectin, and also showed 100-U/mL PGase (polygalacturonase) activity, which catalyzed the hydrolysis of polygalacturonic acid. The Km value for protopectin was 0.215 mg/mL, while the Km value for polygalacturonic acid was 39.09 mg/ml. The PPase/PGase q-value was 2.68, more than 0.5. Therefore the enzyme was considered a novel pectinase and classified as protopectinase.     

兔肺血管紧张素转换酶的分离提取及特性研究

采用DEAE SepharoseFF离子交换色谱分离方法从兔肺中分离提取血管紧张素转换酶 (ACE)。采用马尿酰组氨酰亮氨酸 (HHL)作为底物测得其酶活力为 1 95U /mg ,最适温度和 pH值分别为 3 7℃和 8 3。经与商品兔肺ACE进行对照实验表明 ,分离得到的兔肺ACE与商品兔肺ACE的性质完全相同     

茯苓粗多糖的提取及开发前景展望

研究了微波辅助酶法提取茯苓多糖的工艺。通过优化条件、设计正交试验,确定最佳提取工艺条件为:以水为浸提剂,微波功率750 w,料液比1∶30,提取时间15 min。对茯苓多糖的应用前景进行了展望。研究表明茯苓多糖具有较大的开发价值。     

牛骨蛋白酶解制取肽钙的研究进展

文章首先简要介绍了牛骨的成分,重点对羟基磷灰石和胶原蛋白的结构进行了描述;接着对常用酶的作用位点以及酶解的研究现状进行了详细介绍。通过对氨基酸螯合钙和多肽螯合钙的螯合和吸收机制进行比较,确定多肽螯合钙的优越性和研究的必要性,并对目前多肽螯合钙的结构分析方法,不同分子量多肽钙的分离以及多肽钙产品的研发情况进行了详细概括。     

榆干离褶伞溶栓酶的纯化及酶学性质研究

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纯化一种溶栓酶,并探讨各种因子对榆干离褶伞溶栓酶活性的影响,观察溶栓酶对纤维蛋白及纤维蛋白原的水解特征和酰胺水解活性。实验结果表明,其分子质量约为50 ku,该酶的最适pH和最适温度为pH 6.0,35℃,并对tPA底物S-2288最敏感。Mn~(2+)和Mg~(2+)激活酶活性,而Cu~(2+),EDTA对酶活有抑制作用。该酶不仅直接水解纤维蛋白,还可以水解纤维蛋白原。榆干离褶伞很有潜力成为治疗血栓的新的药物来源。