Heat-resistant Enzyme Research Articles

Dynamic Interplay between Microbiota Shifts and Differential Metabolites during Dairy Processing and Storage.

Due to the intricate complexity of the original microbiota, residual heat-resistant enzymes, and chemical components, identifying the essential factors that affect dairy quality using traditional methods is challenging. In this study, raw milk, pasteurized milk, and ultra-heat-treated (UHT) milk samples were collectively analyzed using metagenomic next-generation sequencing (mNGS), high-throughput liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). The results revealed that raw milk and its corresponding heated dairy products exhibited different trends in terms of microbiota shifts and metabolite changes during storage. Via the analysis of differences in microbiota and correlation analysis of the microorganisms present in differential metabolites in refrigerated pasteurized milk, the top three differential microorganisms with increased abundance, Microbacterium (p < 0.01), unclassified Actinomycetia class (p < 0.05), and Micrococcus (p < 0.01), were detected; these were highly correlated with certain metabolites in pasteurized milk (r > 0.8). This indicated that these genera were the main proliferating microorganisms and were the primary genera involved in the metabolism of pasteurized milk during refrigeration-based storage. Microorganisms with decreased abundance were classified into two categories based on correlation analysis with certain metabolites. It was speculated that the heat-resistant enzyme system of a group of microorganisms with high correlation (r > 0.8), such as Pseudomonas and Acinetobacter, was the main factor causing milk spoilage and that the group with lower correlation (r < 0.3) had a lower impact on the storage process of pasteurized dairy products. By comparing the metabolic pathway results based on metagenomic and metabolite annotation, it was proposed that protein degradation may be associated with microbial growth, whereas lipid degradation may be linked to raw milk's initial heat-resistant enzymes. By leveraging the synergy of metagenomics and metabolomics, the interacting factors determining the quality evolution of dairy products were systematically investigated, providing a novel perspective for controlling dairy processing and storage effectively.

Impact of Thermo-sonication on Pectin Methylesterase (PME) Inactivation &amp; Cloud Stability of Nagpur Mandarin Juice

Nagpur mandarin is well known globally for its excellent nutritional benefits and flavour. These all characteristics get affected during storage due to a lack of knowledge &amp; processing techniques. One of the most important parameters which decide the quality of fruit juice during storage is cloud stability, which gets affected during storage due to the action of an enzyme named pectin methylesterase. Pectin methylesterase enzyme (PME) is a heat-resistant enzyme that impacts the juice quality by decreasing the cloud stability. Thermosonication is a technique to reduce the PME activity in juice by utilizing the principle of heat and ultrasound. Therefore, an experiment was carried out to inactivate the PME utilizing the ultrasound combined with higher temperature known as thermosensation on Nagpur mandarin (Citrus reticulata Blanco) juice. This study was initiated to calculate the effect of thermosensation treatment on the pectin methylesterase enzyme (PME) activity along with the effect of thermosensation on cloud stability in comparison with various thermal pasteurization treatments. Juice samples were thermosonicated at temperatures of 50 ℃, 55 ℃, 60 ℃, and 63 ℃ for 25, 20, 15 and 10 min respectively at a constant frequency of 40 kHz. Juice samples were also thermally pasteurized at 65 ℃, 75 ℃, 85 ℃, and 95 ℃ for 80, 60, 40, and 20 sec. Samples were analyzed after treatment at 0 days and 7 days of refrigerated storage at 4 ℃. Result indicated that thermosensation at 63 ℃ for 10 min at 40 kHz frequency showed much reduced PME activity with inactivation of 72.56% over control whereas thermal pasteurization at 95 ℃ showed inactivation of 69.31%. The same treatment also showed maximum cloud activity as compared to control and thermally pasteurized juice samples. Hence thermosensation can be used as an alternative processing technique for the inactivation of PME and cloud stability.

Identificação de atividade deteriorante e do gene apr na microbiota isolada de leite cru em Caxias, MA

Dentre as bactérias contaminantes, as psicrotróficas destacam-se como as principais responsáveis pela deterioração do leite, pois são capazes de se multiplicar em temperaturas de refrigeração. O gene apr carreia a informação para a produção da metaloprotease alcalina, uma enzima termoresistente capaz de degradar as proteínas do leite. Objetivou-se investigar, por meio de métodos biomoleculares, a presença do gene apr e o potencial deteriorante de bactérias proteolíticas isoladas do leite cru em uma microrregião do Maranhão. Foram selecionadas 112 amostras de leite cru, coletadas de fevereiro a junho de 2018 em propriedades leiteiras das microrregiões de São João do Sóter e Caxias, MA. As análises microbiológicas foram realizadas no Instituto Federal do Maranhão (IFMA), Campus Caxias, e os ensaios biomoleculares no Laboratório de Inspeção de Produtos de Origem Animal (LIPOA) da Universidade Estadual de Londrina (UEL). Foram realizados testes para classificação de proteólise, lipólise e potencial acidificante em temperatura ótima (36 °C/2 dias) e atividade psicrotrófica (7 °C/10 dias). Foram achados 10 (9%) isolados positivos para o gene apr, todos pertencentes ao gênero Pseudomonas, corroborando com outras pesquisas no estado que apontam para o grande potencial deteriorante das bactérias encontradas na localidade. Desta forma, assim como em outras regiões do Brasil, o leite cru refrigerado produzido no estado do Maranhão precisa ser obtido de forma higiênica evitando contaminação com microrganismos deteriorantes que possam comprometer a integridade e a qualidade do leite fluido e derivados destinados ao comércio.

Microwave inactivation of red beet (Beta vulgaris L. var. conditiva) peroxidase and polyphenoloxidase and the effect of radiation on vegetable tissue quality

Abstract The inactivation of polyphenoloxidase (PPO) and peroxidase (POX) in red beet by traditional and microwave (MW) blanching was studied. Microwave heating effects on color and texture were also studied. Red beet subjected to MW blanching for 5 min at 100–200 W resulted in large weight losses accompanied by a high degree of shrinking. POX was the more heat resistant enzyme. The 90% destruction ( D value) of the activity of both enzymes could be achieved only at 200 W within the 5 min frame employed for the tests. When the samples were immersed in water and both the food sample and the water were submitted to MW exposure at 250–450 W or variable power with a maximum at 935 W, shrinking was avoided. The D value at 90 °C (reference; D T ref ) and z could be determined after time–temperature corrections, and it was observed that, in general, D T ref values for POX were smaller than for PPO. The microwave treatment (maximum power: 935 W) designed to provide a similar temperature profile to the one observed for traditional blanching (immersion in water at 90 °C), showed the smallest D T ref value for POX inactivation. All treatments reduced elastic characteristics and changed the color of the tissues showing a shift to blue mainly in the case of microwave processes.

PURIFICATION AND CHARACTERIZATION OF A CHITINASE FROM PEANUT (ARACHIS HYPOGAEA L.)

ABSTRACT A chitinase was isolated from peanut (Arachis hypogaea L.) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, and high-performance liquid chromatography on POROS 20 HQ. There was a 133-fold increase in specific activity of purified chitinase compared with that of the crude extract. The protein exhibited a molecular mass of 34.4 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and nonreducing conditions, indicating that it is a monomeric protein. The isoelectric point was 5.1 by isoelectric focusing electrophoresis. Optimal pH activity was 5.4 and optimal temperature was 40–50C. The enzyme was stable below 55C, but was rapidly inactivated when incubated at temperatures above 60C. These results demonstrated that the purified protein was a kind of relatively thermostable chitinase from the peanut seeds. PRACTICAL APPLICATIONS This paper describes the isolation and characterization of a chitinase from Arachis hypogaea L. seeds. Chitinases are listed as the class of pathogenesis-related proteins, and they have become a popular research field because of their resistance to plant disease. The research we are presenting should help in understanding chitinase functions in plant defense mechanisms. A relatively thermostable enzyme newly obtained, which was stable below 55C, would certainly be significant for its utilization both in enzymology and in plant defense. In particular, a relatively heat-resistant enzyme from A. hypogaea L. seeds would certainly be helpful for elaborating strategies for constitutive expression of chitinase genes and their manipulation and general utilization in plant defense mechanisms through more recent techniques of genetic manipulation.

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin is a heat resistant enzyme, catalyzing the oxidation of coelenterazine

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin (BFP-aq) was prepared and determined to be a heat resistant enzyme, catalyzing the luminescent oxidation of coelenterazine (luciferin) with molecular oxygen as a general luciferase. After treatment with excess ethylenediaminetetraacetic acid to remove Ca 2+ from BFP-aq, the blue fluorescence shifted to a greenish fluorescence. This greenish fluorescent protein (gFP-aq) was identified as a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1. By incubation with coelenterazine in the absence of reducing reagents, gFP-aq was converted to aequorin at 25 °C. BFP-aq and gFP-aq possessing both fluorescence and luminescence activities may work as novel reporter proteins.

THERMAL DESIGN PROCESS FOR ACIDIFIED CANNED PAPAYA (Carica papaya L.) PULP

ABSTRACT Previous results showed that the heat resistant portion of pectinesterase in the acidified papaya pulp var. “formosa” presented greater thermal resistance than Cl. pasteurianum. Based on this, and determined F7.8C77C, a value of 12.3 min was calculated using the heat resistant portion of pectinesterase as the target for the process, and applying 1.7 decimal reduction of the activity of the heat resistant enzyme (98% inactivation). Using Shiga (1976) an empirical relationship and a heating time of 12.9 min in water bath at 97C was established for the process. Three processing tests were carried out under these conditions using 6 cans per test. The processing values varied from 12.33 to 12.75 min at 77C. Statistical analysis showed no significant difference between tests and among cans as well as test at the 0.05 and 0.01 α level. After processing the cans, gelatinization, activity of remaining enzyme and micro‐organism growth were not detected. This shows that the process was able to inactivate the heat resistant portion of pectinesterase and kill the natural micro‐flora present on the pulp. By considering the initial variation of the pectinesterase activity in the pulp and F7.8C77C ‐ value of 16.7 min, a safety factor for the process was applied. This process was equivalent to 15 min of heating in water bath at 97C and corresponded to 2.3 decimal reductions in activity of heat resistant pectinesterase. An inoculation pack study was performed to verify the microbiological safety of such process. Each can of papaya pulp was inoculated with 1 mL of a 104 spore/mL suspension of Cl. pasteurianum. Swelling of the cans, growth after subculturing and pectinesterase were not detected after incubation, showing that the pulp could be safely processed. The storage test showed no significant changes in the color, flavor, aroma and texture of the canned acidified papaya pulp.

Muconolactone isomerase of the 3-oxoadipate pathway catalyzes dechlorination of 5-chloro-substituted muconolactones.

An enzyme of Alcaligenes eutrophus JMP 134 which catalyzes dechlorination of (4R, 5R)- and (4R,5S)-5-chloro-3-methyl- and (4R, 5S)-5-chloromuconolactone of principally 3-methyl-trans-, 3-methyl-cis-dienelactone and cis-dienelactone, respectively, was purified to homogeneity. The enzyme was identified as muconolactone isomerase on the basis of its high activity with muconolactone and on its high degree of sequence similarity with previously described muconolactone isomerases. Molecular mass determinations of the highly hydrophobic and heat-resistant enzyme indicated a decameric structure involving a single 10.100-kDa subunit similar to that of muconolactone isomerase of Pseudomonas putida. Kinetic analysis showed cooperative effects between the subunits during conversion of (4R, 5S)-5-chloro-3 -methylmuconolactone. (4R, 5S)-5-chloromuconolactone was the preferred substrate, over the natural substrate (4S)-muconolactone. The (4S, 5S)-structure was found to be an inhibitor of (4R, 5R)-5-chloro-3-methylmuconolactone transformation. Methylsubstitution of the substrate results in a higher affinity for the enzyme, but a drastically lower velocity, resulting in a lower specificity constant.

耐熱性酵素の新しい利用

耐熱性酵素の新しい利用

Use of bispecific hybrid antibodies for the development of a homogeneous enzyme immunoassay

Hybrid bispecific monoclonal antibodies reacting with carcinoembryonal antigen (CEA) and with the E. coli enzyme β-galactosidase (GZ) were produced by fusion of hybridomas or chemical linkage of half-antibodies. Since the original anti-GZ antibody used in these experiments was capable of protecting GZ from thermal denaturation, it was possible, by hybridizing it with two different non-competitive anti-CEA antibodies, to design a homogeneous enzyme immunoassay for quantitation of CEA. In fact, a mathematical analysis of the reaction indicates that, under appropriate concentrations of the reactants, circular complexes can be formed which contain the two hybrid antibodies, the GZ enzyme and the CEA antigen. The stability of these complexes can be expected to be substantially greater than that of the more labile CEA-free GZ-antibody complexes, prompting a significant increase in the amount of enzyme molecules which are bound to antibody and are consequently protected from thermal denaturation. These expectations were supported by experimental results: under appropriate conditions, heat-resistant enzyme activity was indeed proportional to concentration of CEA in the range up to 75 ng/ml. As predicted by theory, however, in the presence of excess CEA — in fact at CEA concentrations which are higher than those of possible clinical relevance — circular complexes tended to open up, leading to a marked prozone effect.

Localization of the thermosensitive X-prolyl dipeptidyl aminopeptidase in the vacuolar membrane of Saccharomyces cerevisiae

Most of the X-prolyl dipeptidyl aminopeptidase activity of Saccharomyces cerevisiae was found to be associated with purified vacuolar membranes (specific activity approx. 75-times higher than in the protoplast lysate). The tonoplast-bound enzyme is thermosensitive. Another heat-resistant enzyme was found in the protoplast lysate. The tonoplast-bound thermosensitive enzyme shows an apparent K m of 0.06 mM against L-alanyl-L-prolyl- p-nitroanilide while the heat-resistant enzyme shows an apparent K m of 0.4 mM against the same substrate.

Thermal Inactivation of Peroxidase in Relation to Quality of Frozen Cauliflower (var. Indian Snowball)

A blanching procedure is described for cauliflower (var. Indian Snowball) based on inactivation of the heat resistant enzyme, peroxidase. The thermal inactivation time (F) of the enzyme was 1.0min at 96.3°C and the corresponding decimal reduction time (D) was 0.38min with z values (temperature required to change F or D values by a factor of 10) of 13.6 and 13.3C°, respectively, thus giving a 2.63 D process for adequate blanching. Based on the quality evaluation of frozen cauliflower stored up to 10 moat-18°C, a 1.5 D process was recommended for blanching prior to freezing. Blanching times for achieving 1.5 and 3.0 D processes were computed for cauliflower florets having different stem thicknesses.

Determination of Thermal Process Schedule for Acidified Papaya

ABSTRACTThe pH of papaya varies from 4.2‐5.65 and is required to be lowered to enable processing at atmospheric pressure. A thermal process has been evolved on the basis of inactivation of pectinesterase (PE), the heat resistant enzyme naturally present. The values for thermal inactivation of PE in pulp were F26.8212= 1.04 and D27212= 0.39 at pH 4.0. In the syrup homogenate of 20% TSS, the values were F27.6207.3= 1.00 and D29207.3= 0.53 at PH 3.8, and F27.2212= 1.23 and D27.5212= 0.8 at pH. 4.0. These F values in syrup homogenate were equivalent to 1.88D at pH 3.8 and 1.5 3D at pH 4.0. In commercial canning, a 2.5D process at pH 3.8 and 2.0D process at pH 4.0 which are equivalent to the F values of 1.33 and 1.31, respectively, are recomended. Using these F values, process times for different processing conditions were calculated by graphical method and compared with values found by three different procedures of the formula method. The thermal process, thus evolved on the basis of enzyme inactivation, resulted in a safe and acceptable product.

Quantitative studies on acid phosphatase in developing rat bones and teeth during hypervitaminosis D

The following differences were found between bone and tooth buds with regard to the acid p-nitrophenyl phosphatase activity in whole homogenates and extracts: (a) most of the enzyme in a KCl (0.15 M) homogenate of bone was in a 6000 g min sediment but was extracted with urea, while most of the activity in tooth buds was extracted with KCl and was not affected by urea; (b) the activity in bone was stimulated by both sodium tartrate and sodium chloride in vitro, while the activity in tooth bud was markedly inhibited by sodium tartrate and unaffected by sodium chloride; (c) the activity in bone, but not in tooth buds, was completely inhibited by fluoride in vitro and relatively resistant to heat; and (d) the enzyme activity in bone, but not in teeth, was increased markedly during hypervitaminosis D. The tissue-specific effects of inhibitors in vitro and of vitamin D in vivo are in agreement with previous histochemical studies with α-naphthyl phosphate as substrate. It is concluded that most of the p-nitrophenyl phosphatase activity in bone is due to a tartrate-resistant and heat-resistant enzyme which is different from the tartrate-sensitive and heat-labile enzyme responsible for most of the activity in the odontogenic cells.

PURIFICATION AND PROPERTIES OF L-ALANINE DEHYDROGENASE FROM VEGETATIVE CELLS OF BACILLUS CEREUS.

McCormick, Neil G. (University of Wisconsin, Madison), and Harlyn O. Halvorson. Purification and properties of l-alanine dehydrogenase from vegetative cells of Bacillus cereus. J. Bacteriol. 87:68-74. 1964.-The l-alanine dehydrogenase from vegetative cells of Bacillus cereus strain T has been purified approximately 200-fold. The enzyme has a molecular weight of 248,000 and a turnover number of 80,000 moles of substrate per min per mole of enzyme. The Michaelis constants for the substrates and the equilibrium constant for the reaction catalyzed by this enzyme are in close agreement with reported values for other l-alanine dehydrogenases. The kinetic properties of the enzyme purified from vegetative cells are identical to those of the enzyme isolated from spores of the same organism, but differ with respect to relative heat stability. Whereas spores contain a heat-resistant enzyme, vegetative cells contain, in addition, a heat-sensitive enzyme. No evidence was found to support the hypothesis that a molecular conversion type of phenomenon plays a role in the appearance of spore enzyme.

Studies on the spores of aerobic bacteria. II. The properties of an extracted heat-stable enzyme

1. 1. Spores of Bacillus terminalis were found to contain alanine racemase activity which was only 10 % inactivated when held at 80 ° for 2 hr. On the other hand, the alanine racemase associated with the vegetative cells of this organism was almost completely inactivated after 15 min. at this temperature. 2. 2. Cell-free sonic extracts of these spores were found to contain alanine racemase which was also active after 2 hr. of heating at 80 °. 3. 3. Fractionation of crude sonic extracts by ammonium sulfate precipitation or high-speed centrifugation revealed that the heat-resistant enzyme was associated with particulates. 4. 4. Further sonic oscillation of collected spore particles solubilized some of the racemase. The solubilized enzyme was heat-sensitive. 5. 5. Particulate alanine racemase was resistant to pepsin inactivation as well as to heat denaturation. The soluble, heat-sensitive form was, however, readily inactivated by pepsin. 6. 6. The enzyme required pyridoxal phosphate for full activity after 60 % resolution by dialysis.

The Decomposition of Yeast Nucleic Acid by a Heat Resistant Enzyme

The Decomposition of Yeast Nucleic Acid by a Heat Resistant Enzyme

Effect of a Heat-Resistant Enzyme Upon the Antigenicity of Pneumococci

Rabbits immunized by the intr a cutaneous injection of heat-killed encapsulated pneumococci fail to develop the type-specific carbohydrate antibodies which result from the intravenous injection of the same antigen.1 An attempt was made to follow the fate of pneumococci injected into the skin, in the hope of determining the cause of this lack of type-specific antigenic response.Heat-killed cells of pneumococcus Type I were injected at several sites into the skin of a rabbit. The injected areas were excised at different intervals of time and films made from the tissue fragments were stained by the Gram technic. There was, of course, a pronounced polymorphonuclear infiltration at the site of injection. The pneumococci were seen to undergo a process of extracellular digestion which began within 24 hours after injection and was completed in 4 to 5 days; many bacteria became Gram negative before being engulfed by the leucocytes. These observations suggested that leucocytes produce ferments capable of attacking ...