The precipitation of peptides and proteins by lignosulphonic acids

The precipitation of peptide- and protein-lignosulphonic acid complexes can be demonstrated and studied in agar plates in the form of precipitation zones. The effects on the precipitates caused by variations in incubation temperature, incubation time, concentration of the reagents and in pH in the mixture of reagents, are described, and the nature of the peptide-lignosulphonic acid bond is discussed. The central lysis zones which appeared in some precipitation zones were found to be probably caused by excess of lignosulphonic acids. The possibility of developing the agar precipitation method described as a direct micro quantitative procedure for the determination of certain lignosulphonic acids in aqueous solution is suggested.     

The effects of gamma irradiation on some properties of two Aeromonas proteinases

The proteinases A and B of Aeromonas liquefaciens and proteinase B of Aeromonas salmonicida have, in the crude and purified state, been exposed to various doses of γ-irradiation from a Co⁶⁰ source, and the D₃₇ values indicate that the proteinase A is the most resistant.Casein was shown to have a marked protective effect on both proteinases during the irradiation, and while solutions of the purified enzymes were inactivated by doses usually used for food pasteurization, the crude enzymes, or solutions of purified enzymes to which casein was added, required doses usually used for food sterilization before being inactivated. Only minor effects of the environmental pH were observed. The antigenic properties of the enzymes seemed to be qualitatively unchanged in solutions exposed to 150 krad as observed using the casein precipitation inhibition test, and the irradiated proteinases were also inhibited by the naturally occurring proteinase inhibitors in the immune sera. The enzymoserological properties were not influenced by the changes in electrophoretic migration which were demonstrated by the zymogram technique. These proteinases are suitable as models for the examination of the physical properties of food spoiling enzymes and also for taxonomical work.Keyword: gammairradiation, preservation, Aeromonas, proteinases, enzymes, CP -test, zymograms     

Acid phosphatase in cheese

The combined action of proteinases/peptidases and acid phosphatases on phosphorylated proteins/peptides could be important for cheese quality. It seems that acid phosphatase activity in cheese originates from milk and microorganisms. Bovine milk contains more than one acid phosphatase, which are distributed between the cream and skim milk. The properties of two enzymes isolated from butter milk and skim milk are different. In general, acid phosphatase, especially that from starter organisms is very heat stable. The dephosphorylation of casein and phosphopeptides is of interest in cheese ripening. It has been suggested that acid phosphatases from both skim milk and lactic acid bacteria contribute to dephosphorylation of phosphopeptides in cheese, which are produced from casein by coagulant, indigenous milk proteinase and microbial proteinases during cheese ripening.     

Toxocara canis: proteinases in perivitelline fluid from hatching eggs

Developing larvae of Toxocara canis may secrete several kinds of enzymes within the egg perivitelline fluid (EPF) prior to and during hatching. In particular, proteinases in EPF could play a role in larval emergence within the host gastrointestinal lumen but its presence and nature is unknown. In this work, proteolytic activities in hatching fluid of T. canis were identified and analysed by substrate gel electrophoresis at different pH values and by using type specific protease inhibitors. Three bands of 91, 68 and 38 kDa showed gelatinolytic activity and all proteinase activity from EPF was of the aspartic-type since it was inhibited by pepstatin A. Interestingly, a significantly higher proteolytic activity was observed at acidic pH (< or =5.5). These data suggest that T. canis developmentally secretes and accumulates in EPF aspartic proteinases with a pH-dependent activity that might help the parasite to take advantage of conditions in the host gastrointestinal microenvironment where egg hatching is induced and executed.     

Proteinase, lipase and amylase activities in the small intestine of pigs suffering from colienterotoxaemia.

The activities of proteinases, lipases, amylases and the activities of proteinase inhibitors, as well as the numbers of Escherichia coli in the contents from the small intestine were examined for pigs suffering from colienterotoxaemia and for healthy pigs. Enzyme activities were determined using an agar diffusion test.Compared with healthy animals the activities of proteinases and amylases in diseased animals were reduced while lipases showed increased activity. In pathologically changed contents showing large numbers of E. coli, proteinases could not be demonstrated; however, proteinase inhibitors were found in these contents. In healthy animals, proteinase inhibitors were not demonstrated in ingesta-con-taining contents.In diseased animals, E. coli were found in large numbers in all parts of the small intestine. In healthy animals, E. coli was demonstrated especially in the posterior part of the small intestine and regularly in small numbers.The possible influence of digestive enzymes, especially proteinases and their inhibitors, on enterotoxins from E. coli is discussed.     

The Effect of Diethylaminoethyl Dextran and Agar Overlay pH on Plaque Formation by Two Plaque-size Variants of Foot-and-Mouth Disease Virus

The effect of diethylaminoethyl (DEAE) dextran and agar overlay medium pH on a small-plaque (SP) and large-plaque (LP) foot-and-mouth disease virus (FMDV), type A, strain 119 (A119) was studied. The SP virus was inhibited under normal agar overlay but the addition of 100, 1,000 and 2,000 µg DEAE dextran/ml of agar overlay permitted plaque development. By using untreated and DEAE dextran-treated agar overlay medium, plaque formation by the SP virus was enhanced when the pH of agar medium was raised to a more alkaline level before overlay. Plaques formed by the LP virus were relatively uninhibited under the regular overlay but were larger in the presence of 1,000 µg DEAE dextran/ml. The enhancement of LP virus plaques occurred at various pH levels and was also inversely related to the hydrogen ion concentration of agar overlays; regular and DEAE dextrantreated alkaline overlays produced larger plaques.     

The effects of peptide-precipitating lignosulphonic acids on the in vitro proteolytic activity of some animal and microbial proteinases

It has been shown that lignosulphonic acids have an inhibitory effect on various animal and microbial proteinases. The activity of 0.005 mg swine trypsin, 0.007 mg bovine a-chymotrypsin and the proteinase activity in 0.004 ml of jejunum content from pig is inhibited 70–75 % by 0.25–0.30 ml of a solution containing 250 diffusion units of peptide-precipitating lignosulphonic acids per 50 µl.The inhibitory effect is discussed in relation to the use of animal foodstuffs containing lignosulphonic acids, and in relation to biochemical reactions taking place in ecosystems in which lignosulphonic acids comprise an important part.     

Comparative studies on the in vitro properties of phytases from various microbial origins.

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60 degrees C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80 degrees C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70 degrees C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40 degrees C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.     

Properties of Brucella canis surface antigens associated with colonial mucoidiness

Rough Brucella strains B. abortus (45/20), B. ovis (1182), wild type B. canis (M+) and a less mucoid (M-) variant possessed cell wall antigens that were extracted by both sodium desoxycholate (SDC) and hot phosphate buffered saline (PBS). The acid precipitability of cell wall surface antigens extracted in SDC from all four strains suggests that these antigenic complexes may be responsible for the bacterial aggregation in broth media at acid pH and for the relatively high colonial viscosity on normal agar media (pH 6.8). The antigens of B. canis (M+) extracted in PBS were serologically related to those from the other 3 strains, but they differed in acid precipitation and hydrophobic characteristics. Differences between the properties of B. canis surface antigens and those of the other rough Brucella may explain the highly mucoid nature of the canine organism when grown on conventional (pH 6.8) media.     

Consequences of calcium interactions with phytate and phytase for poultry and pigs

Despite increasing practical experience and cascades of scientific reports on exogenous microbial phytases, several issues associated with their use remain unresolved because of the ambiguous and, at times, conflicting data that has been generated. One possible cause of these inconsistent outcomes is dietary calcium (Ca) levels, which are mainly derived from limestone. Thus the purpose of this review is to examine Ca interactions with dietary phytate and phytases, particularly exogenous, microbial phytases, and their consequences for poultry and pigs. The polyanionic phytate molecule has a tremendous capacity to chelate cations and form insoluble Ca–phytate complexes, which are refractory to phytase activity. Thus Ca–phytate complex formation along the gastrointestinal tract, where one phytate (IP₆) molecule binds up to five Ca atoms, assumes importance and approximately one third of dietary Ca may be bound to phytate in digesta. Consequently, phytate limits the availability of both P and Ca as a result of insoluble Ca–phytate complex formation, the extent of which is driven by gut pH and molar ratios of the two components. It is accepted that Ca–phytate complexes are mainly formed in the small intestine where they have a substantial negative influence on the efficacy of mucosal phytase. However, exogenous phytases are mainly active in more proximal segments of the gut and lower pH levels, so their efficacy should not be influenced by Ca–phytate complexes in the small intestine. There is, however, data to indicate that Ca and phytate interactions occur under acidic conditions with the formation of soluble and insoluble Ca–phytate species, which could negatively impact on exogenous phytase efficacy. Also, Ca will tend to elevate gut pH because of limestone's very high acid binding capacity, which will favour Ca–phytate interactions and may influence the activity of exogenous phytases depending on their pH activity spectrum. The de novo formation of binary protein–phytate complexes that are refractory to pepsin hydrolysis may be fundamental to the negative impact of phytate on the digestibility of protein/amino acids. However, high dietary Ca levels may disrupt protein–phytate complex formation by interacting with both phytate and protein even at acidic pH levels, thereby influencing the outcomes of phytase amino acid digestibility assays. Finally, it is increasingly necessary to define the Ca and nonphytate-P requirements of pigs and poultry offered phytase-supplemented diets.     

Screening and Identification of the Cellulase-producing Fungus

In order to isolate the strains which could degrade fiber from the straw,the straw samples were inoculated on the potato agar medium and cultured at room temperature.The isolating strains were inoculated on the cellulose-congo red agar medium,the strains were screened that could produce bigger cellulose-decomposing zone.It was identified by morphological characteristics and molecular biological methods,and characterization of cellulase was analyzed preliminarily.The results showed that the fungus was Aspergillus niger,it could grow at room temperature,and the H/C was 7.64.The highest cellulase activity reached 42.18 U/mL when cultivated at 20 ℃ for 5 days.The optimum pH was 7.0 and the optimum reaction temperature was 20 ℃,the relative cellulase activity still retained above 90% at 20 to 40 ℃ or pH 6.0 to 8.0 for 1 h.The Aspergillus niger was a cold-adapted cellulaseproducing strain,it had strongger producing cellulase ability,and was tremendous potential valuable for microbial development.     

产纤维素酶真菌的筛选与鉴定

为从秸秆中分离出能够降解纤维的菌株,试验采取秸秆样品接种马铃薯琼脂培养基,在室温环境下培养,取分离菌接种纤维素刚果红琼脂培养基,筛选能形成较大降解圈的细菌,并进行形态学和分子生物学鉴定,测定其纤维素酶性质。结果表明,经分子生物学鉴定,筛选菌为黑曲霉菌,该菌在室温环境能够生长,菌株降解圈直径(H)与菌落直径(C)的比值(H/C)为7.64。筛选菌在20 ℃环境下培养5 d酶活达到最高值,为42.18 U/mL。纤维素酶最适pH为7.0,最适反应温度为20 ℃,在20~40 ℃或pH 6.0~8.0环境下作用1 h,相对酶活力仍保持在90%以上。综上所述,本试验分离的黑曲霉菌株是低温产纤维素酶菌株,产酶能力较强,具有潜在的开发价值。     

水土保持植物类芦对土壤酸胁迫的形态生理响应

土壤酸度低是限制一些金属矿山废弃地植被恢复的主要因素之一,类芦可在酸度极低的稀土矿废弃地正常生长,其中可能存在对酸胁迫特殊的形态生理响应机制。因此,采用室内土培盆栽模拟胁迫试验,设计不同强度酸胁迫处理(pH: 3.5,4.5,5.0和5.6),测定不同酸胁迫条件下,类芦地上部分和根系生长、生物量、抗氧化酶活性及MDA含量等指标,探讨类芦对土壤酸胁迫的适应策略。结果表明,类芦对土壤酸胁迫有一定的耐性,pH 3.5处理仍能较正常生长;随土壤pH的逐渐增大,类芦地上部分和根系各生长指标整体表现为先增大后减小的变化趋势,弱酸胁迫对类芦根系生长有一定促进作用,pH 4.5处理均达最大值,为类芦最适宜生长的土壤酸度;酸胁迫条件下,类芦可通过加快根直径的生长,增大根系的生物量分配,以适应酸胁迫条件,但酸胁迫对类芦体内抗氧化酶活性具有一定抑制作用。     

Detection and characterization of Theileria sergenti proteinases.

The lysate of Theileria sergenti piroplasms was tested for proteinases using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which substrate was included in gel matrix. Six proteinases of molecular weight 330, 125, 98, 94, 67 and 58 kilodalton (kDa) were detected. From the results of the Triton X-114 phase partition, 330, 125 and 58 kDa proteinases were partitioned into aqueous phase, which indicated that they were not associated with parasite membranes. All these three enzymes were classified into metalloproteinase family because of their sensitivities to metal-ion chelating compounds, ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline. On the other hand, 98 and 94 kDa proteinases were membrane-associated metalloproteinases which were preferentially inhibited by 1,10-phenanthroline. Another metalloproteinase of 67 kDa which was inhibited by EDTA and 1,10-phenanthroline was not associated with parasite membranes. Proteinases of 98 and 94 kDa degraded heat-denatured hemoglobin.     

西藏地区荞麦与玉米混合青贮对发酵品质和微生物多样性的影响

为探究西藏林芝市全株荞麦与全株玉米混合青贮对青贮饲料发酵品质和微生物多样性的影响,分别设定全株荞麦单独青贮组(A)、全株荞麦∶全株玉米=4∶1混合青贮组(B)、全株荞麦∶全株玉米=3∶2混合青贮组(C)、全株荞麦∶全株玉米=2∶3混合青贮组(D)和全株荞麦∶全株玉米=1∶4混合青贮组(E)共5个处理组。分别在青贮第7、14、30和60天时,开窖取样,测定青贮饲料的发酵品质和微生物菌群结构。结果表明,与全株荞麦单独青贮组相比,全株荞麦与全株玉米混合青贮组在一定程度上改变了青贮的发酵品质,混合青贮组降低了青贮饲料的pH值,提高了乳酸含量,且氨态氮/总氮的值和丁酸含量均符合优质青贮饲料的要求量。从微生物菌群结构来看,混合青贮改变了青贮饲料的菌群结构,相比与全株荞麦单独青贮组,混合青贮组提高了厚壁菌门和LAB菌种的丰度,有效地抑制了腐败菌的生长,且这种效果随着全株玉米混合比例越高而越显著。综合考虑全株荞麦利用最大化和发酵品质,建议将全株荞麦和全株玉米以2∶3混合青贮较为适宜。     

DNases in milk and blood sera from different species.

DNases were demonstrated in samples of colostrum and blood serum from man and various domestic animals. The measurable DNase activity recorded was highest in samples from cat and dog and lowest in samples from goat, horse, pig and sheep. In contrast to DNases produced by certain bacteria, these enzymes were thermo-labile and the activity was maximal in the area pH 5.0–5.5.A modification of an agar medium originally described for the demonstration of bacterial DNases was found to be suitable for assays of DNases from colostrum, milk and serum.     

Molecular cloning of two Haemaphysalis longicornis cathepsin L-like cysteine proteinase genes.

Immunological protection of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method to the current use of acaricides which has several limitations. The success of this method is dependent on the identification of key molecules for use as tick vaccine antigens. Proteolytic enzymes are involved in a wide range of cellular processes in eukaryotes such as development regulation and nutrition, thus they can be considered as good target antigens for a tick vaccine. In the present study we used primers designed based on the consensus amino acid motifs flanking the conserved active sites C25 and N175 present in all papain-like cysteine proteinases to amplify by polymerase chain reaction, sequence and characterize two Haemaphysalis longicornis tick cysteine proteinase genes. Based on the nucleotide and deduced amino acid sequences, both genes were identified as members of the cysteine proteinase gene family by presence in their sequences of consensus motifs flanking the conserved active sites C25, H150 and N175 that are present in all papain-like cysteine proteinases. Both genes are about 1.2 kb in size and show high sequence homology predominantly to invertebrate cathepsin L-like cysteine proteinases.     

Proteinases of the cornea and preocular tear film

Maintenance and repair of corneal stromal extracellular matrix (ECM) requires a tightly coordinated balance of ECM synthesis, degradation and remodeling in which proteolytic enzymes (proteinases) perform important functions. There are natural proteinase inhibitors present in preocular tear film (PTF) and cornea simultaneously with proteinases that prevent excessive degradation of normal healthy tissue. Disorders occur when there is an imbalance between proteinases and proteinase inhibitors in favor of the proteinases, causing pathologic degradation of stromal collagen and proteoglycans in the cornea. Two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are of major importance in terms of remodeling and degradation of the corneal stromal collagen. Immunohistochemical studies have shown different origins of MMP-2 and -9. MMP-2 is synthesized by corneal keratocytes and performs a surveillance function in the normal cornea, becoming locally activated to degrade collagen molecules that occasionally become damaged. Alternatively, MMP-9 may be produced by epithelial cells and polymorphonuclear neutrophils following corneal wounding. Because the cornea is in close contact with the preocular tear film (PTF), proteinases have been evaluated in the PTF. In damaged corneas, total proteolytic activity in the tear fluid was found to be significantly increased compared to normal eyes and contralateral eyes. Studies analyzing the proteolytic activity in serial PTF samples during corneal healing led to the following conclusions: ulcerative keratitis in animals is associated with initially high levels of tear film proteolytic activity, which decrease as ulcers heal; proteinase levels in melting ulcers remain elevated leading to rapid progression of the ulcers. The success of medical and surgical treatment of the corneal ulcers is reflected by the proteolytic activity in tears. In animals, successful treatment leads to a rapid reduction in tear film proteolytic activity that corresponds with the improvement in the clinical signs of corneal ulceration. The in vitro effects of various compounds on proteolytic activity in the tear fluid of animals with ulcerative keratitis have been evaluated and their important inhibitory effects have been confirmed. Because these various compounds utilize different mechanisms to inhibit various families of proteinases, a combination of these proteinase inhibitors may be beneficial.     

Studies on Aspergillus Fumigatus; Hydrolysis of Polyamino Acids by Mycelial Filtrate and Chromatographic Fractionation of the Filtrate

Mycelial filtrates from Aspergillus fumigatus (AF), shown to possess haemolytic, toxic, casein precipitating, and protein hydrolyzing activity, hydrolyzed poly-L-lysine and poly-L-glutamine in the pH range 4.6—5.3. Incipient activity against poly-L-lysin was observed at pH 9. Owing to spontaneous hydrolysis of the polyamino acid at pH > 10, no activity optimum could be traced.Gel filtration of mycelial filtrate on Sephadex G-75 or G-100 columns offered no definite information whether the protein hydrolyzing activity, using haemoglobin as substrate, at the optimum pH values, 2.9, 4.6 and 10, shows the activity of a single enzyme with more than 1 pH optimum or of more than 1 enzyme active at different pH values. Certain results of the investigations seem to indicate that the protein hydrolyzing activity at pH 2.9 was not caused by enzymes identical with the enzyme (s) causing the protein hydrolyzing activity at pH 4.6 or pH 10.Casein precipitating and protein hydrolyzing activity occurred, following gel filtration on a Sephadex G-100 column, in identical fractions whereas neither haemolysin nor toxin could be found in samples of 0.5 ml fraction solution from any of the fractions after filtration on Sephadex G-75 or G-100 columns.By using antiserum to a crude filtrate from a homologous AF strain it could be shown, applying immuno-electrophoresis, that dialyzed mycelial filtrate contained 8 precipitating antigens whereas proteinase purified by gel filtration and displaying protein hydrolyzing activity at pH 2.9, pH 4.6 and pH 10 contained 4 such antigens.