Application of Scharer's quantitative method for the determination of residual alkaline phosphatase activity in standard Minas / Aplicação do método modificado de Scharer para a determinação quantitativa da atividade de fosfatase alcalina residual em queijo minas padrão

Milk pasteurization is a critical issue in the dairy industry, and failures in this process can affect final product safety. Scharer's enzymatic method is still traditionally used to verify pasteurization efficiency compliance, and it is based on screening for residual alkaline phosphatase in milk. Although several methods are used to quantify enzymatic activity to assess milk pasteurization efficiency, there is a small amount of published data regarding the use of these methods to quantify alkaline phosphatase in cheese. In this study, the Scharer's modified method was used to determine the levels of residual alkaline phosphatase in standard minas cheese, before and after 20 days of ripening. The cheeses were made using raw or pasteurized milk with the addition of different concentrations of raw milk (0; 0.05%; 0.10%; 0.20%; and 0.50%). In the fresh cheese samples, the method showed a sensitivity of only 0.50% with the addition of raw milk to the pasteurized milk used to make cheese. In addition, levels of up 0.20% of raw milk in pasteurized milk, the concentrations of phenol was inferior to 1μg phenol/g of dairy product which is the preconized indicator value for adequate pasteurization.

A pasteurização do leite é um ponto crítico na indústria de laticínios, e falhas nessa etapa comprometem a segurança do produto. O método enzimático de Scharer é tradicionalmente utilizado na verificação da eficiência da pasteurização e baseia-se na pesquisa da atividade de fosfatase alcalina residual em leite. Embora vários métodos estejam disponíveis para avaliar a eficiência da pasteurização, há um número reduzido de dados publicados baseados na quantificação da atividade da fosfatase alcalina em queijo. Neste estudo, o método modificado de Scharer foi utilizado para determinar os níveis de fosfatase alcalina residual em queijo minas padrão, antes e após 20 dias de maturação. Os queijos foram feitos com leite cru ou com leite pasteurizado com adição de diferentes concentrações de leite cru (0, 0,05%, 0,10%, 0,20% e 0,50%). Nas amostras de queijo fresco, o método apresentou sensibilidade apenas com 0,50% de adição de leite cru ao leite pasteurizado utilizado na fabricação de queijo. Em níveis de adição de até 0,20% de leite cru no leite pasteurizado, as concentrações de fenol se mostraram inferiores a 1μg de fenol/g de produto lácteo, que é o valor preconizado como indicador de pasteurização adequada.

Kinetic behaviour of calf intestinal alkaline phosphatase with pNPP.

The hydrolysis of p-nitrophenyl phosphate (pNPP) by calf intestinal alkaline phosphatase (CIAP) was investigated with respect to kinetic parameters such as Vmax, Km and Kcat under varying pH, buffers, substrate concentration, temperature and period of incubation. Highest activity was obtained with Tris-HCl at pH 11, while in the case of glycine-NaOH buffer the peak activity was recorded at pH 9.5. The enzyme showed the following kinetic characteristics with pNPP in 50 mM Tris-HCl at pH 11 and 100 mM glycine-NaOH at pH 9.5 at an incubation temperature of 37°C: Vmax, 3.12 and 1.6 µmoles min-1 unit-1; Km, 7.6 × 10-4 M and 4 × 10-4 M; and Kcat, 82.98 s-1 and 42.55 s-1, respectively. CIAP displayed a high temperature optimum of 45°C at pH 11. The kinetic behaviour of the enzyme under different parameters suggested that the enzyme might undergo subtle conformational changes in response to the buffers displaying unique characteristics. Bioprecipitation of Cu2+ from 50 ppm of CuCl2 solution was studied where 64.3% of precipitation was obtained. Pi generated from CIAP-mediated hydrolysis of pNPP was found to bind with copper and precipitated as copper-phosphate. Thus, CIAP could be used as a test candidate in bioremediation of heavy metals from industrial wastes through generation of metal-phosphate complexes.

Impact of transgenic cotton varieties on activity of enzymes in their rhizosphere.

The impact of five Bacillus thuringiensis (Bt) cotton varieties and their respective isogenic non-Bt(NBt) isolines (ANKUR-2534, MECH-6304, RCH-317, ANKUR-651 and MECH-6301) was assessed on the key soil enzymes i.e., dehydrogenase, alkaline phosphatase and urease in their rhizosphere at four growth stages of the crop, namely vegetative, flowering, bolling and harvesting. These varieties were grown on farmer’s field in villages 22 miles and 24 miles of Ganganagar District of Rajasthan State in India. Results showed that dehydrogenase, alkaline phosphatase and urease activities were higher in rhizosphere of Bt isolines as compared to NBt isolines of all the varieties. Except phosphatase, differences in dehydrogenase and urease activities in rhizosphere of Bt and NBt isolines of all five varieties were significant (P<0.05). Maximum enhancement in the three enzymes activities was observed in MECH-6304 Bt isoline rhizosphere. Maximum and minimum activities of dehydrogenase and urease were observed in MECH-6304 and RCH-317 Bt isolines, respectively, whereas phosphatase activity was maximum and minimum in MECH-6304 and ANKUR-651 Bt isolines, respectively. Maximum dehydrogenase and urease activities were observed at boll formation and minimum at flowering and harvesting stage, respectively, while maximum phosphatase activity was observed at vegetative stage and minimum at harvesting stage. In conclusion, all the studied Bt isolines of cotton varieties showed no adverse effect on dehydrogenase, alkaline phosphatase and urease activities in the rhizosphere.

Identification of digestive hydrolases in the larval midgut of the camel nasal bot fly cephalopina titilla tor clark [diptera: Oestridae]

Activities of digestive hydrolases associated with midgut of the third instar larva of Cephalopina titillator were investigated. Based on the hydrolysis of synthetic substrates and optimum pH, it was found that C. titillator midgut contains trypsin-like [optimum pH, 9], chymotrypsin esterase-like [optimum pH, 8], carboxypeptidase A and B [optimum pH at 8.5 and 7 respectively], alkaline- and acidphosphatase [optimum pH at 9 and 5 respectively] and membrane bound leucine aminopeptidase [optimum pH, 8]. An acid proteinase activity was detected, by the ability to hydrolyze acid denaturated haemoglobin; and it seems to be close to pepsin than cathepsin-like enzyme. It has a maximum activity at pH 3.5. alpha-Glucosidase activity, and was also identified [optimum pH at 6] in the midgut, and seems to be membrane bound

Study of the new human molehydatiform alkaline phosphatase

Alkaline Phosphatase [EC: 3.1.3.1] is synthesized by kidney, liver, bone, Intestine and placenta. This enzyme is a glycoprotein and dimmer 4 Zn+2 and Mn+2 in each dimmer. It hydrolyzes mono ester phosphate to organic compound and phosphate_in alkaline medium. The purpose of this research is to compare this enzyme with placental alkaline phosphatase. Human Molehydatiform was purified by folds of precipitation of bybutanol, acetone, Amoniumm sulphate, Sephadex G200, affinity chromatography and preparative electrophoresis. Human Molehydatiform was purified 611.8 times. We obtained specific activity, optimum temperature and optimum Ph equaling to 611.8 U/mg, 40 centigrade degrees and 10.4 respectively. Purified Human Molehydatiform Alkalie phosphatase is different from Human placental Alkaline phosphatase due to optimum pH and optimum temperature

Kinetic studies and electrophoretic separation of alkaline phosphatase isoenzyme from cancerous patients

The activity of sALP was determined by the calorimetric method King Armistrong in serum of 303 cancerous patients and 42 normal healthy controls. The results show that the ALP activity was increased in all types of cancer tissues, however ALP activity show a significant increase only in bone, liver and pancreas tissues. The increase of ALP activity could be used as tumor marker for bone, liver and pancreas tissues. The increase in sALP activity could be used as tumor marker for bone and liver cancer and to detect metastasis to the organ. No significant differences of sALP activity were found between males and females. The activity was increased with the stage of development of the disease. The kinetic studies for normal healthy human were measured at a substrate concentration 10 mM, Km 2.7 mM, temperature 370c and pH,o. The electrophoretic studies show that one band of ALP in serum of norma subjects, as compared to cancerous patients where several bands were detected in cancer patients; the intensity of band color varied with type; age and stage of disease

Alkaline phoshatase, tumor necrosis factor-alpha and matrix metalloproteinase-8 in crevicular fluid from peri-implantitis versus chronic periodontitis patients

Peri-implantitis is an inflammatory reaction affecting the tissues surrounding osseointegrated dental implants resulting in loss of supporting bone. Recent advances in the understanding of biologic events involved in the pothogenesis of periodontitis indicating that bone mediators e.g. tumor necrosis factor-alpha [TNF-alpha], alkaline phosphatase [ALP] and matrix metalloproteinase-8 [MMP-8] may also be operating in the pathogenesis of peri-implantitis. This study aimed to explore whether pro-inflammatory mediator TNF-alpha and markers of bone loss; ALP and MMP-8 in per-implant crevicular fluid [PICF] provide a diagnostic information as to the status of the implant. The present study evaluated 11 implants in patients having peri-implantitis and 12 without implantitis as compared to 12 patients with chronic periodontitis. The clinical assessment for all patient groups included pocket depth [PD], plaque index [PI] and gingival index [GI]. There were significant differences [p<0.05] in PI, PD and GI in peri-implantitis and periodontitis patient groups as compared to healthy implant group, while there were non significant difference between per-implantitis and periodontitis patient groups. ALP, MMP-8 and TNF-alpha were measured in gingival crevicular fluid [GCF] and PICF 2 years postoperatively. The ALP activity and MMP-8 concentration were significantly higher in periodontitis and peri-implantitis patients than healthy implant group [p<0.01, and p<0.05, respectively]. There were no statistically significant differences TNF-alpha concentration between the three study groups. There were no statistically significant differences in MMP-8 concentration and ALP activity between periodontitis group and patients with per-implantitis. The ALP activity showed a significant positive correlation with GI and PD [p<0.01]. In conclusion, the present results might suggest that ALP and MMP-8 in PICF has a possible role as a markers of peri-implantitis

Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30ºC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the Km and Hill coefficient values were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the Km and Hill coefficient values were 0.44 mM and 0.97, respectively), ß-glycerol phosphate (the Km and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the Km and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg2+, Zn2+ and Tris-HCl buffer, and is inhibited by Be2+, histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70ºC(half-life of 19.0 min, k = 0.036 min-1) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min-1) in the same experiment

Reconstitution of denatured E. coli alkaline phosphatase with E. coli ribosome.

E. coli alkaline phosphatase was denatured by physical/chemical means. In vitro reconstitution of this denatured enzyme was assisted by 70S E. coli ribosome, as shown by the recovery of its catalytic competence. Almost total recovery of activity of the totally inactivated enzyme was obtained in presence of equimolar concentration of 70S ribosome at 50 degrees C.

Diagnóstico bioquimico clínico de miopatias congénitas: distrofia muscular de Duchene (DMD) y de Becker / Biochemical clinic diagnosis of congenital myopathies: Duchenne's and Becker's muscular dystrophies

Las distrofias musculares tipo Duchene y tipo Becker son enfermedades vinculadas al cromosoma X, con debilidad y alteraciones degenerativas catabólicas de los músculos esqueléticos. Existe consenso en que el efecto genético asociado a ambas enfermedades puede ser detectado en las células musculares así como en una variedad de células no musculares. Se ha investigado la conducta de la fosfatasa alcalina de la membrana de los etitrocitos en paciente con distrofia muscular tipo Duchene, en mujeres portadoreas y en controles sanos. Esta enzima alostérica es útil para detectar alteraciones entre la membana y la enzima en ella incluída. Kinéticas comparativas con coeficientes de Hill de 2,19 ñ 0,21; 1,71 ñ 0,15 y 1,54 ñ 0,16 fueron obtenidos mediante la inhibición con fluoruros, en testigo de control, mujeres portadoras y pacientes con Duchene, respectivamente, con diferencias significativas que convalidan su alcance. Nuestra observación viene en apoyo de las alteraciones de las membranas celulares previamente descritas en ambas distrofias musculares mencionadas, extendiendo su verificación a mujeres portadoras. Tomando en consideración que las alteraciones de la membrana eritrocítica también están presentes en mujeres portadoras del acusador distrófico, la determinación del coeficiente de Hill en la mujer en riesgo, es sugerida como prueba útil para la detección de tales portadoras