Antibacterial activity of proteins extracted from the pulp of wild edible fruit of Bromelia pinguin L.

Bromelia pinguin L. is a natural source of bioactive compounds. The main purpose of this research was to isolate and characterize bioactive proteins from its fruit. B. pinguin proteins were fractionated by gel filtration chromatography, and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antibacterial activity of the proteins was analyzed against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, and the enzymatic activity was evaluated by protease activity and trypsin inhibitions assays. Protein fraction obtained by gel filtration chromatography exhibited antibacterial activity against E. coli (minimum inhibitory concentration [MIC] 0.3492 mg/mL) and S. aureus (MIC 0.6845 mg/mL). The proteolytic activity of the fraction was 0.985 U_cas/mL. The substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay detected protease inhibitors with molecular weights of 43 and 74 kDa. Antibacterial studies of E.coli and S. aureus were determined by comparing the protein fraction with different antibiotics. The antibacterial activity of proteins extracted from the pulp of the fruit of Bromelia pinguin L. could be related to the presence of enzymes, protease inhibitors and peptides.     

Preparation of whey protein hydrolysates with ACE-inhibitory activity using cysteine peptidases from Bromelia hieronymi Mez. (Bromeliaceae)

A proteolytic extract from fruits of Bromelia hieronymi was used to hydrolyse bovine whey proteins. The peptide profile obtained exhibited a gradual fading of the main whey proteins and a decrease in the content of hydrophobic peptides. The 180-min hydrolysate showed a hydrolysis degree of 15.2% and inhibited the angiotensin-converting enzyme (ACE), showing an IC₅₀ of 0.17 mg/mL. By bioinformatics analysis, several theoretical sequences of possible ACE-inhibitory peptides were deduced. These results showed that proteolytic extracts from B. hieronymi can be used to prepare whey hydrolysates that would serve as a potential bioactive ingredient of functional foods.     

Intracellular proteases of Thermoactinomyces vulgaris. 2. Biochemical characterization of proteases in cell extracts

The intracellular proteases (IP) of the cellular extract of Thermoactinomyces vulgaris are characterized as to the biochemical properties compared with the corresponding extracellular proteases (EP). According to that, the storage stability and the temperature and pH behaviour (optimum, stability) of both of the proteases are identical. Nevertheless, differences were detected between IP and EP after the action of several effectors and different substrates. As could be seen after a column chromatographic separation of the IP of the cellular extract, it is composed of at least 3 proteases, two of them are serine proteases which can be inhibited moreover unspecifically by p-chloromercuri benzoate. The purified proteases (IP) are very instable and therefore not yet characterized in detail.     

Chromatographic methods of mackerel proteases isolation

A possibility is shown of producing acidic and alkaline proteases from water fraction obtained during comprehensive processing of hydrobionts. The proteases are obtained by the affinity and ligand-exchange chromatography methods, without resorting to a non-technological stage of enzyme precipitation by ammonia sulphate or organic solvents. The level of specific activity thus achieved for acidic mackerel proteases is 300 u.a./g, for alkaline mackerel proteases 630 u.a./g.     

牛乳中微生物酶的分离及危害研究

牛乳中存在的耐热芽孢杆菌和某些假单胞菌能够分泌蛋白水解酶类。采用(NH4)2SO4沉淀，高速离心的方法对原料乳中微生物产酶进行分离提取，在366m波长处比色，结果证实有一定的酶活存在。通过分析酶与UHT灭菌乳质量的关系，发现酶能使乳蛋白发生水解，酸度增大，并出现沉淀现象。     

毛霉蛋白酶在腐乳成熟中的作用

采用SDS—PAGE与ESI—MS相结合的方法研究了毛霉蛋白酶和Alcalase蛋白酶对SPI的酶促降解作用，研究结果表明，Alcalase是一种典型的内切型蛋白酶，它能在1h内催化大豆蛋白降解为分子量10000D以下的肽段，但降解产物容易相互聚合而沉淀，毛霉蛋白酶则具有较强的肽酶活性，能够以大豆蛋白或多肽为底物酶促降解生成多量的水溶性肽，为后期嗜盐菌的生长提供了可供利用的速效氮素营养，毛霉蛋白酶还具有消除苦味、形成鲜味物质的作用，与腐乳风味形成有关。     

Characterization of ginger proteases and their potential as a rennin replacement

BACKGROUND: Ginger rhizome (Zingiber officinale Roscoe) contains ginger proteases and has proteolytic activity. Ginger proteases have been used for tenderizing meat but rarely for milk clotting. The purpose of this study was to purify ginger proteases and to research their biochemical characteristics. RESULTS: The milk clotting activity (MCA) and proteolytic activity (PA) of the proteases was stable after storage at 4 °C for 24 h. The MCA and PA of fresh ginger juice with 0.2% L ‐ascorbic acid remained stable for 6 days at 4 °C. When under storage at ?80 °C for 2 months, the MCA and PA of the fresh ginger juice and acetone precipitate were still high. Two peaks with protease activity were purified from a DEAE FF ion‐exchange column; the specific activity (units mg^?1 protein) of the MCA (MCSA) and PA (PSA) for the first peak was significantly higher than the second peak (P < 0.05). The protease activity of the ginger proteases was significantly inhibited by E‐64, leupeptin, and iodoacetic acid. Zymography results showed that two protease fractions purified from ginger juice with 62 and 82 kDa had a higher PA against α‐ and β‐casein than against κ‐casein. CONCLUSION: The ascorbic acid addition significantly stabilized the MCA and PA of ginger proteases. The protease inhibition test suggested that ginger proteases belonged to the cysteine type. The biochemical characteristics of ginger protease described in this paper can provide useful information for making new milk curd products. Copyright © 2009 Society of Chemical Industry     

Production of proteases by psychrotrophic microorganisms.

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20 h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52 degrees C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.     

蛋白酶的保健应用

蛋白酶与我们的日常生活密不可分,其作用涉及到细胞生命循环、代谢调节和身体防卫的许多方面,表现在：水解食物中的蛋白质,辅助分解衰竭细胞,清除入侵的微生物或蛋白质并产生可激发创伤恢复的功能性肽,以确保生命代谢循环的正常运行。西方国家利用包括蛋白酶在内的水解酶类保健至少有70年以上的历史,而我国尚未将一些主要的可大量生产的天然蛋白酶类列入天然保健品或膳食添加剂的原料名单中。本文简略介绍了几种主要蛋白酶的功能特性、应用安全性、国内外市场发展等方面的情况。蛋白酶作为国产天然保健品开发似有良好前景。     

The effect of legume protease inhibitors on native milk and bacterial proteases

Protease inhibitors from legume seed extracts (soybean, cowpea and marama beans) and purified soybean protease inhibitor were evaluated with regards to their abilities to inhibit proteases produced by important milk contaminating bacteria, i.e. Bacillus spp. and Pseudomonas spp., and native milk protease, plasmin. Although heat treatment is the most common mean of inactivating enzymes, some heat-stable enzymes can survive the ultra-high temperature (UHT) processing of milk and cause sensory and consistency defects during storage at room temperature. The legume protease inhibitors reduced the activity of plasmin and proteases produced by Bacillus spp. by up to 94% and 97%, respectively, while it showed low inhibitory activity towards Pseudomonas fluorescens proteases (19%) in a buffer system. The protease inhibitors reduced the activity of plasmin (41%) and Bacillus proteases (50%) in UHT milk, however to a lesser extent as compared to inhibition in the buffer system; while it had little or no effect on proteases form Pseudomonas spp. Legume protease inhibitors show great potential in preventing or reducing proteolytic activity of Bacillus proteases and plasmin and may be exploited in various applications where these proteases cause sensory or consistency defects in the product.     

蛋白酶对羊毛作用的探讨

为了全面提高羊毛品质,选用诺LanL和定劳沙WSLnew蛋白酶,采用H2O2氧化预处理,单一蛋白酶以及H2O2-蛋白酶二步法对羊毛进行改性处理.结果表明经双氧水预处理后,蛋白酶更容易向纤维内部扩散,并对其发生作用;羊毛经这2种蛋白酶处理后能发生Allworden现象并可部分消除其表面壁障,使上染率提高.     

Preliminary investigation of the properties of somatic cell proteases

Effect of the plasmin inhibitor 6-amino-n-hexanoic acid on somatic cell proteases (equivalent to 2.3 X 10(6) cells/ml) was determined using a model system of casein micelles dispersed in Jenness/Koops buffer (1.5% wt/vol). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to quantitate casein. There was no effect of 120 mM 6-amino-n-hexanoic acid on casein proteolysis by somatic cell proteases. Molecular weights of casein proteolysis products produced by somatic cell proteases were different from those produced by plasmin. Quantitative and qualitative analyses of pasteurized mastitic milk by SDS-PAGE indicated that a portion of the somatic cell proteolytic activity survived pasteurization. Because 6-amino-n-hexanoic acid does not inhibit somatic cell proteases, it can be used to establish the relative contribution of somatic cell proteases and plasmin to total proteolytic activity in mastitic milk.     

Autodegradation of the extracellular proteases of Brevibacterium linens ATCC 9172

Brevibacterium linens ATCC 9172 produces multiple forms of extracellular proteolytic enzymes as shown by polyacrylamide gel electrophoresis (PAGE) and activity staining. Four main bands with strong proteolytic activity, showing proteins with molecular weights of 280, 220, 130 and 43 kDa, were distinguished from several bands of lower activity. The formation of smaller entities on incubation at 38°C from the isolated main species of proteases was demonstrated, showing specific sequences in autodegradation. The cell wall-associated proteases were completely released by incubation of the cells at 50°C for 2 h and showed a similar isoenzyme pattern. There is evidence that the multiplicity of proteases is a result of aggregation from subunits and of autocatalytic degradation. The enzymes were identified as serine proteases by specific inhibition.     

Fluorescein thiocarbamoyl-kappa-casein assay for the specific testing of milk-clotting proteases

Milk-clotting proteases, which are widely used in the cheese-making industry, are enzymes that use soluble caseins as their preferential substrates. Here, we propose a modification to a method previously described for the specific determination of milk-clotting proteases by using κ-casein labeled with fluorescein isothiocyanate as substrate. Validation of the modified method was confirmed using natural bacterial, fungal, plant, and animal milk-clotting proteases, as well as a milk-clotting enzyme of recombinant origin. The new modified method described here allowed specific quantification of the activity of milk-clotting proteases in a very sensitive way and permitted determination of the appropriate kinetic parameters of all the enzymes tested, consistent with their origin and degree of purity.     

Detergent alkaline proteases: enzymatic properties, genes, and crystal structures

Subtilisin-like serine proteases from bacilli have been used in various industrial fields worldwide, particularly in the production of laundry and automatic dishwashing detergents. They belong to family A of the subtilase superfamily, which is composed of three clans, namely, true subtilisins, high-alkaline proteases, and intracellular proteases. We succeeded in the large-scale production of a high-alkaline protease (M-protease) from alkaliphilic Bacillus clausii KSM-K16, and the enzyme has been introduced into compact heavy-duty laundry detergents. We have also succeeded in the industrial-scale production of a new alkaline protease, KP-43, which was originally resistant to chemical oxidants and to surfactants, produced by alkaliphilic Bacillus sp. strain KSM-KP43 and have incorporated it into laundry detergents. KP-43 and related proteases form a new clan, oxidatively stable proteases, in subtilase family A. In this review, we describe the enzymatic properties, gene sequences, and crystal structures of M-protease, KP-43, and related enzymes.     

Purification, characterization, and milk coagulating properties of ginger proteases

Ginger proteases are used as milk coagulants in making a Chinese traditional milk product (Jiangzhinai or Jiangzhuangnai), suggesting their potential as a source of rennet substitute that might be applicable in the modern dairy industry. In this study, ginger proteases were extracted from fresh ginger rhizome by using phosphate buffer and subsequently purified by ion exchange chromatography. Ginger proteases, all with a molecular weight around 31 kDa, were found to exist in 3 forms with isoelectric point values around 5.58, 5.40, and 5.22, respectively. These enzymes had very similar biochemical behavior, exhibiting optimal proteolytic activity from 40 to 60°C and maximum milk clotting activity at 70°C. They were capable of hydrolyzing isolated α_S1-, β-, and κ-casein, of which α_S1-casein was most susceptible to the enzyme; κ-casein was hydrolyzed with a higher specificity than α_S1- and β-casein. In addition, the ginger proteases exhibited a similar affinity for κ-casein and higher specificity with increasing temperature. Gel electrophoresis and mass spectra indicated that Ala90-Glu91 and His102-Leu103 of κ-casein were the preferred target bonds of ginger proteases. The milk clotting activity, affinity, and specificity toward κ-casein showed that ginger protease is a promising rennet-like protease that could be used in manufacturing cheese and oriental-style dairy foods.     

Effects of high protease-activity flours and commercial proteases on cookie quality

The effects of high-protease activity flour (HPAF) on cookie quality were investigated comparing two proteases (Protease 1 and Protease 2). The commercial proteases and HPAF were added to wheat flours (cvs. Gerek and Gün) with different gluten quality at levels of comparable protease activity. Electrophoresis was used to determine the hydrolyzing effects of the proteases on gluten proteins. Cookies were prepared with wire-cut formulation and quality parameters (spread ratio and color values) were determined. Electrophoresis results showed that degradative effects of Protease 1 and Protease 2 were more evident on gliadins and glutenins, respectively. Protease 2, added to cookie formulations for both wheat samples, gave the highest spread ratios. For the strong wheat cultivar (Gün) spread ratio values of the cookies supplemented with HPAF were comparable to those supplemented with Protease 2.     

蛋白酶对羊毛机织物改性的可行性分析

分别利用诺和LanL、WSLnew及其复合酶对羊毛机织物进行改性处理,经正交实验得出:使用双氧水预处理后,将诺和LanL与WSLnew复合使用,处理羊毛机织物可得到优良的改性性能,经弱酸艳蓝RAW染色的上染速率明显提高,强力保留率可达87%左右.     

Heat-induced tenderisation of meat by endogenous carboxyl proteases

The rôle of carboxyl proteases in tenderising meat was investigated by injecting the inhibitors, pepstatin and EPNP, into pre-rigor muscle. The increase in shear force values induced by these inhibitors provided a minimum estimate of the extent to which endogenous carboxyl proteases normally tenderise meat at 60°C.Gel electrophoresis showed that connectin was hydrolysed to a greater extent than other muscle proteins at this temperature and that breakdown of connectin was inhibited by pepstatin and EPNP. Thus it is likely that, when intact, connectin may contribute to the strength of cooked meat.     

Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin.

Production of heat resistant proteases by psychrotrophs growing in milk, resistance of such proteases to ultrahigh temperature treatments and action of these enzymes on milk were studied. All of the psychrotrophs obtained from raw milk produced proteases that survived 149 C for 10s. Seventy to ninety percent of the raw milk samples contained psychrotrophs capable of producing heat resistant proteases. The protease chosen as a model was resistant to heat treatments at 110 to 150 C, and the inactivation parameters suggested that thermal destruction of heat resistant proteases would damage the milk severely. The casein content and pH of normal milk were suitable for protease action, and the protease was quite active at normal and elevated room temperatures. The protease rapidly spoiled sterile milk with the development of bitter flavor, clearing, or coagulation; and the susceptibility of sterile milk to protease increased during storage of the milk.