草鱼消化道粗蛋白酶的部分性质

用丙酮脱脂然后用磷酸盐缓冲溶液从草鱼肠道中提取一种主要含有酸性蛋白酶和碱性蛋白酶的粗蛋白酶。该粗酶水解血红蛋白的最适pH=3～4，水解酪蛋白的最适pH值有2个，分别为pH4．4和PH10．3。水解牛血红蛋白的最适作用温度为37℃，水解酪蛋白的最适作用温度为45℃。酸性蛋白酶在50℃保温30min后只有大约20％的最初活性被保留，而要使碱性蛋白酶丧失80％的最初活力需要在60℃保温30min。     

鱿鱼墨黑色素酶解法提取工艺优化及其紫外、红外光谱特征分析

以鱿鱼墨水解度为指标,分别测定木瓜蛋白酶、中性蛋白酶、酸性蛋白酶、胃蛋白酶和胰蛋白酶对鱿鱼墨水解效果,选用水解能力最强的胃蛋白酶为试验用酶,测定酶解反应pH值、反应时间、反应温度和加酶量对鱿鱼墨水解度的影响。在单因素试验基础上进行L9(34)正交试验优化得到胃蛋白酶水解鱿鱼墨的最佳酶解条件为酶解反应pH1.5、酶解温度37℃、酶解反应时间5h、加酶量1.5%。在最优酶解反应条件下,胃蛋白酶水解鱿鱼墨的水解度大于13.50%。紫外-可见光谱分析显示酶解法制备的鱿鱼墨黑色素在220nm处有特征吸收波长,红外光谱中的3384.54cm-1和1617.35cm-1处出现吲哚环结构归属的强吸收峰。     

鱿鱼蛋白抗菌液及其美拉德反应产物制备及抑菌作用研究

目的 制备抗菌型鱿鱼蛋白水解液,实现鱿鱼资源的高值化利用。方法 以鱿鱼蛋白(鱿鱼头和加工碎肉)为原料,经风味蛋白酶、胰蛋白酶、胃蛋白酶、碱性蛋白酶和中性蛋白酶水解,测定各水解液对鱿鱼腐败菌(specific spoilage organisms of squid after end storage at room temperature,SE-SSOs)的抑菌性,选择合适蛋白酶水解鱿鱼蛋白,在最适酶解温度和酶解pH下,研究加酶量和酶解时间对水解液抑菌活性影响,制备鱿鱼蛋白抗菌液,通过美拉德反应提高鱿鱼蛋白抗菌液对SE-SSOs的抑菌活性,探讨抑菌模式。结果 胃蛋白酶水解鱿鱼蛋白所得水解液对SE-SSOs的抑菌效果强于其他4种蛋白酶,在胃蛋白酶最适pH 2.0和酶解温度37 ℃下,胃蛋白酶添加量500 U.g_-1^和酶解2 h制备了鱿鱼蛋白抗菌液(Squid protein antibacterial hydrolysate,SAH),SAH中分子量6500 Da左右和低于1000 Da肽组分的相对含量达到46%左右。在SAH中分别添加1%和3%质量体积比(m/v)葡萄糖、果糖和壳聚糖,在120 ℃油浴下加热30 min进行美拉德反应,其中添加3%果糖的SAH美拉德反应产物(记作SAH-3Fru MRPs)对SE-SSOs抑菌作用强于SAH和其他美拉德产物。扫描电镜结果显示SE-SSOs经SAH和SAH-3Fru MRPs作用,其中一些杆菌发生严重膜损伤,而球菌的质膜相对保持较完整。结论 胃蛋白酶水解法适用于制备抗菌型鱿鱼蛋白水解液,通过美拉德反应能进一步提高水解液的抑菌作用,SAH和SAH-3Fru MRPs均可以通过膜损伤方式抑制SE-SSOs中特定菌。因此,SAH及SAH-3Fru MRPs有进一步开发应用前景。     

酶法水解鱿鱼皮制备多肽工艺的研究

分析了阿根廷鱿鱼皮的一般化学组成,并以其为原料,研究了酶的种类、加酶量、pH、温度及酶解时间对水解度(DH)的影响。采用正交实验对酶解工艺进行了优化,研究结果表明,复合蛋白酶水解鱿鱼皮制备多肽工艺的最适条件为:加酶量14000u/g、pH8.5、酶解温度50℃、酶解时间4h。在此条件下水解度可达20.7%。     

菌源性弹性蛋白酶特性的研究

试验通过硫酸铵对发酵液的分段盐析得到粗酶，并对粗酶的基本性质进行了一定的研究。结果表明：酶反应的最适温度为50℃，最适pH为7．4；酶在37℃以下热稳定性良好；重金属对酶活性有不同程度的抑制作用；弹性蛋白对弹性蛋白酶具有较强的亲和吸附作用，弹性蛋白酶有优先水解弹性蛋白的能力。     

天然有机物HFS对酸性蛋白酶酶活的影响

利用酶制剂处理食品原料,进行生产,历史悠久,酶的最大应用对象也是食品工业。在现代食品工业中,酶的应用几乎涉及食品加工的各个领域。随着酶制剂的应用日益广泛,经济效益显著。蛋白酶是水解蛋白质肽链的一类酶的总称,而酸性蛋白酶适宜在酸性条件下水解动植物蛋白质,通过内切和外切作用将蛋白质水解为小肽和氨基酸。本文探讨了天然有机物-HFS对537酸性蛋白酶酶活的影响,测定了该酶的最适反应条件(最适温度为55℃,最适pH值为3.0),考察并探讨了天然有机物-HFS对537酸性蛋白酶最适反应条件的影响。试验结果表明天然有机物-HFS对537酸性蛋白酶具有较好的激活作用,可有效提高酶活约8%,对该酶的最适反应条件无影响,也不改变最适条件曲线的基本变化规律。     

超声波对中性蛋白酶酶解谷朊粉影响的研究

以活性谷朊粉为原料,研究超声作用及超声功率对不同的底物浓度、酶浓度、反应温度、pH经中性蛋白酶水解的谷朊粉水解度的影响。结果表明,超声作用既不改变酶解反应的最适温度和pH,也不改变水解度与水解温度、pH、底物浓度、酶浓度之间关系曲线的变化趋势,但超声作用可明显提高水解度;200W的超声波对中性蛋白酶水解谷朊粉具有明显的促进作用。     

微波处理对酶解谷朊粉的影响

以谷朊粉为原料,研究了微波预处理对中性蛋白酶水解谷朊粉水解度的影响。结果表明,微波作用不改变酶解反应的最适温度和pH,也不改变水解度与水解温度、pH、底物浓度、酶浓度之间关系曲线的变化趋势,但使谷朊粉酶解后的水解度提高了。在微波功率540W,作用时间120s的预处理后,中性蛋白酶水解谷朊粉的最佳工艺条件为:酶加量3%E/S,反应温度40℃,pH 8.0,底物量8%,水解时间2h,水解度为31.58%。     

蛋白酶水解豆渣制备大豆肽的研究

以大豆豆渣为原料,分别采用木瓜蛋白酶、碱性蛋白酶、中性蛋白酶对大豆豆渣进行水解制备大豆肽.研究了3种蛋白酶在不同温度、pH值、水解时间、酶浓度下水解大豆豆渣的最适酶反应条件,以及水解产物对酸豆乳发酵的影响.结果得出,实验豆渣蛋白质含量为10.4 g/(100 g);木瓜蛋白酶的最适水解条件是底物质量分数5％,酶浓度20 000 U/g,水解温度为50℃,pH7,水解6h,最大水解度可达87.31％;碱性蛋白酶的最适水解条件是底物质量分数5％,酶浓度15 000 U/g,水解温度为55℃,pH9,水解3h,最大水解度可达91.23％;中性蛋白酶的最适水解条件是底物质量分数5％,酶浓度15 000 U/g,水解温度为45℃,pH7,水解8h后达到最大水解度,最大水解度可达81.42％.     

秘鲁鱿鱼皮明胶抗氧化肽的制备及其分子质量分布

目的:基于鱿鱼皮明胶肽的抗氧化活性,优化制备工艺,探究其分子质量分布。方法:分别使用4种蛋白酶水解鱿鱼皮明胶,通过测定体外抗氧化活性来确定最适水解用酶。以DPPH自由基清除率为指标,利用单因素及响应面法优化明胶抗氧化肽的制备工艺,通过Tricine-SDS-PAGE及Sephadex G-25凝胶过滤层析测定明胶肽的分子质量分布。结果:碱性蛋白酶为最适水解用酶,最适水解温度55℃,酶添加量7 000 u/g(E/S),时间109 min,底物明胶质量分数6%,pH 6.66。此条件下明胶肽的DPPH自由基清除率达93.18%。90%明胶肽的分子质量介于1.4~10 ku之间。     

CYSTEINE PROTEINASE ACTIVITY IN JUMBO SQUID (DOSIDICUS GIGAS) HEPATOPANCREAS EXTRACTS

Jumbo squid specimens were captured, dissected, and the hepatopancreas was freeze dried for the extraction of proteolytic enzymes. An autolysis experiment conducted at 25C showed two peaks with maximum proteolysis at pH 3 and 5. The proteinase activity of the extract was measured using azocasein, BANA, Z‐PAAFC and Z‐AAAFC substrates. Activity of the extract with azocasein (pH 5) had a maximum at 55C and increased threefold with the inclusion of cysteine or DTT. The proteinase activity remained at least at 60% of the original after 45 h at 4C in the pH range of 3–8. Activity was inhibited 70–85% when extracts were treated with cysteine proteinase specific inhibitors. The proteinases extracted from jumbo squid hepatopancreas are mainly of the cysteine type and have significant activity towards a cathepsin L specific substrate. The understanding of proteinases from this tissue could have implications for quality control of jumbo squid food products.     

Autolysis and the endogenous proteinases characterised in beardless barb (Anematichthys apogon) muscle

Beardless barb is a common fish species used in fermentation of fish paste Ka-pi-plaa. Autolytic profile of beardless barb muscle showed the maximum autolysis was at 50 °C, at both acidic and alkaline pH values. With augmentation concentration of NaCl, autolytic activity slightly decreased. Endogenous proteinases isolated from fish muscle in crude extract forms were also characterised. The acidic proteinases had optimum activity at pH 3.0 and 50°C, and they showed higher proteolytic activity than the alkaline proteinases which were optimally active at pH 9.0 and 50 °C. Proteinases in peak at pH 3.0 were inhibited by pepstatin A, but those in peak at pH 9.0 were highly inhibited by PMSF, TLCK and soybean trypsin inhibitor, suggesting that both aspartic and serine proteinases were existed in beardless barb muscle. The proteinases were stable in pH range of 2.0-5.0 but unstable at the temperatures higher than 40 °C. NaCl suppressed the proteolytic activity, ATP activated the proteinase activity, while CaCl₂, MgCl₂ and CoCl₂ exhibited no influence on the activity. The results implied that cathepsin D is the predominant proteinase responsible for autolysis in beardless barb. The findings were useful to improve the processing and qualities of Ka-pi-plaa product using beardless barb as raw material.     

Identification of a cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas as cathepsin L

A cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas was partially purified by a two step procedure involving ammonium sulfate precipitation and gel filtration chromatography and further by SDS–PAGE. The molecular weight of the proteinase was 24 kDa determined by SDS–PAGE and 23.7 kDa with mass spectrometry. The activity had an optimum pH of 4.5 and optimum temperature of 55 °C under the assay for cathepsin L specific synthetic substrate Z-PAAFC. The cathepsin B and H specific synthetic substrates Z-AAAFC and H-AMC did not show any hydrolysis with the partially purified enzyme. Peptide mapping of trypsin digests of the 24 kDa band from SDS–PAGE showed the squid cysteine proteinase was homologous to cathepsin L from different animal sources. The activity of the partially purified fraction with the cathepsin L specific substrate Z-PAAFC was inhibited 75–89% by enzyme inhibitors specific for cysteine proteinases but was also significantly inhibited by serine and aspartate proteinase inhibitors.     

Autolysis of Squid Mantle Muscle Protein as Affected by Storage Conditions and Inhibitors

Autolysis of squid mantle muscle was investigated under various conditions using muscle homogenate as a model system. Cleavage of myosin into heavy and light meromyosin was prominent during autolysis. Storage conditions such as NaCl concentration and temperature affected not only the rate of autolysis but also the products formed. Conditions for maximal autolysis were: NaCl concentration 0.3M, pH 7 and temperature 40°C. Autolysis was well inhibited by ethylenediaminetetra acetic acid and Na-pyrophosphate at <25°C and by phenyhnethylsulfonyl fluoride and chymotrypsin inhibitor extracted from potato tuber at >35°C suggesting two different types of proteinases were involved.     

Heat-activated proteolysis in lizardfish (Saurida tumbil) muscle

Autolysis of lizardfish mince and washed mince during heating at elevated temperatures was studied. Higher degradation of myosin heavy chain (MHC) was generally observed in mince, compared to washed mince. The highest autolysis was observed at 65 and 60 °C for mince and washed mince, respectively. Autolysis was extended as the incubation time increased by the result of the decrease in MHC band intensity on SDS-PAGE and the increase in TCA-soluble peptides. Autolysis of washed mince was markedly inhibited by E-64 and soybean trypsin inhibitor, suggesting that myofibril-associated proteinases were both cysteine and serine proteinases. Sarcoplasmic proteinase was characterized to be heat-activated alkaline proteinase, which had the optimal pH and temperature of 8.0 and 65 °C, respectively. The preteinase was inhibited by E-64 and activated by reducing agents, which was one of the characteristics of cysteine proteinase. Therefore, endogenous sarcoplasmic and myofibril-associated proteinases play an important role in degradation of myofibrillar proteins of lizardfish during heat-induced gelation, which results in gel weakening.     

Autolysis and biochemical properties of endogenous proteinases in Japanese sandfish (Arctoscopus japonicus)

Autolysis of Japanese sandfish ( Arctoscopus japonicus ) was studied. The highest autolysis was observed at 60 °C. Optimal pH for autolysis was found at both acidic and alkaline pH values. Autolytic activity decreased with increasing NaCl concentration. Further assay of the extract from Japanese sandfish exhibited the maximum activity at 55 °C and pH 9.5. The Japanese sandfish proteinases were markedly inhibited by pepstatin A at pH 3.5, while soybean trypsin inhibitor, PMSF and TLCK inhibited activities of proteinases at pH 9.5, suggesting Japanese sandfish proteinases were both aspartic and serine proteinases. The extracts were stable to heat treatment up to 50 °C for 15 min and still remained high relative activity after incubated at 50 °C for up to 8 h. Maximum stability of the extracts was observed between pH 6 and 11 and no changes of activity were found after incubation at pH 9.5 for up to 8 h. Activity of Japanese sandfish extract continuously decreased as NaCl concentration increased (0–30%), while activity increased as CaCl₂ concentration increased. The results suggest that major proteinases in Japanese sandfish were trypsin-like serine proteinases.     

Endogenous proteinases in true sardine (Sardinops melanostictus)

Characterisation of the autolytic profile of true sardine (Sardinops melanostictus) indicated the involvement of heat-activated proteinases, active at both acidic and alkaline pH values. Autolytic activity decreased with increasing NaCl concentration (0–30%). When crude proteolytic activity in true sardine was studied, two activity peaks were observed, at pH 3.5 and 9.0. Crude proteinase extracts exhibited the highest activity at 55 °C and 60 °C when assayed at pH 3.5 and 9.0, respectively. The pH 3.5 peak activity was effectively inhibited by pepstatin A, while the pH 9.0 peak activity was mostly inhibited by soybean trypsin inhibitor, PMSF and TLCK, suggesting that the various proteinases were present in true sardine. The enzymes were stable for up to 8 h at 55 °C. The activities were also stable at a pH range of 2.0–4.0 and still retained high activity toward hemoglobin after incubation at pH 3.5 for 8 h. Activities of the crude extract continuously decreased as NaCl concentration increased. ATP showed no effect on enzyme activity, while CaCl₂ and MgCl₂ activated the proteinase activity. The results implied that pepsin is a predominant enzyme responsible for autolysis in true sardine during fish sauce fermentation.     

The effect of proteinase inhibitors in food protein hydrolysis by digestive proteinases of white shrimp (Penaeus vannamei) larvae

This study describes the digestive capacities of 10‐day‐old white shrimp postlarvae (PL10) and how some inhibitors of proteinases affect the digestion of protein in aquafeeds by using in vitro hydrolysis techniques. Biochemical data showed eight active proteinases in the PL10 hepatopancreas extract. Enzymes belong to the metallo‐ and serine‐proteinase classes. The effect of inhibitors present in protein ingredients and aquafeeds on PL10 proteinases showed that ovalbumin alone and in commercial microcapsules yielded a significant inhibition in proteolytic activity of PL10 hepatopancreas enzymes. The capacity of PL10 proteinases to hydrolyse the protein fraction within different sources and microcapsules was demonstrated by two in vitro approaches, the pH‐stat (degree of hydrolysis, DH) and electrophoresis (coefficient of protein degradation, CPD). It was shown that PL10 proteinases hydrolyse, in different extent protein sources and microcapsules. Casein, cuttlefish meal and feeds containing these ingredients are quickly hydrolysed. By contrast, ovalbumin and microcapsules containing ovalbumin are not hydrolysed. Copyright © 2006 Society of Chemical Industry     

CHARACTERIZATION OF PROTEINASE CLASSES IN LANGOSTILLA (PLEURONCODES PLANIPES) AND CRAYFISH (PACIFASTACUS ASTACUS) EXTRACTS

Proteinases (EC 3.4.21-24) in langostilla extract and crayfish hepatopancreas were classified using site specific effectors or chelators for either serine, cysteine, aspartic or metallo classes. Azocasein hydrolysis by langostilla and crayfish preparations was inhibited 50% and 40%, respectively, when assayed in the presence of serine proteinase inhibitor, phenylmethylsulfonyl fluoride. A similar degree of inhibition was observed with a trypsin inhibitor. However, no inhibition occurred with a chymotrypsin inhibitor. Less inhibition of azocasein hydrolysis, i.e., 20% and 14%, respectively, resulted with 1,10 phenanthroline. Inhibitors for cysteine and aspartic proteinases did not reduce the azocasein hydrolysis activities significantly. The amidase activity, with N-benzoyl-DL-Arg-p-nitroanilide as substrate was more sensitive, in both extracts, to serine proteinase inhibitors than the azocasein hydrolase activity. Results showed the presence of serine proteinases, i.e., trypsin but not chymotrypsin, as the major component and some minor activity of metallo dependent proteinases in the decapods extracts. Zymograms obtained after SDS-PAGE showed a dozen proteinases in each extract. Some of them were inhibited by a serine proteinase inhibitor and two to three were inhibited by 1,10 phenanthroline, supporting the results of the test tube proteinase activity assays. Furthermore, the reported technique for zymograms allowed direct comparison between extracts and provided information about their composition and the molecular weight of the targeted enzymes.     

Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation

Abstract: Cell‐associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation were extracted and characterized. Proteinases were effectively released when washed cells were incubated in 0.3 mg/mL lysozyme in 50 mM Tris‐maleate (pH 7) at 37 °C for 2 h. Major cell‐associated proteinases exhibited molecular mass of 17, 32, and 65 kDa, but only a 32‐kDa proteinase showed strong amidolytic activity toward Suc‐Ala‐Ala‐Pro‐Phe‐AMC. Activity of all cell‐associated proteinases was completely inhibited by phenylmethanesulfonyl fluoride, indicating a characteristic of serine proteinase. In addition, a 65‐kDa serine proteinase was also inhibited by ethylenediaminetetraacetic acid, implying a metal‐dependent characteristic. Optimum activity toward a synthetic peptide substrate was at 50 °C and pH 8 and 11. Proteinases with molecular mass of 17 and 32 kDa exhibited caseinolytic activity at 25% NaCl and activity based on a synthetic peptide substrate increased with NaCl concentrations up to 25%, suggesting their role in hydrolyzing proteins at high salt concentrations. This is the first report of liberated cell‐associated proteinases from a moderate halophile, Virgibacillus sp. Practical Application: The cell‐associated proteinases could be extracted from Virgibacillus sp. SK 33 using lysozyme. The extracted enzyme could be applied to hydrolyze food proteins at NaCl content as high as 25%. In addition, this study demonstrated that not only extracellular but also cell‐associated proteinases are key factors contributing to protein‐degrading ability at high salt environment of Virgibacillus sp. SK 33.