Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases.

SummaryCrude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources.LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercapto-ethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lyso-phosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet’s response to several agonists.

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Thermal Inactivation of Susceptible and Multiantimicrobial-Resistant Salmonella Strains Grown in the Absence or Presence of Glucose

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.4123-4128.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 4123-4128 ◽

Cited By ~ 32

Author(s):

R. T. Bacon ◽

J. R. Ransom ◽

J. N. Sofos ◽

P. A. Kendall ◽

K. E. Belk ◽

...

Keyword(s):

Heat Stress ◽

Protective Effect ◽

Thermal Tolerance ◽

Thermal Inactivation ◽

Survival Curves ◽

Acid Adaptation ◽

Circulating Water ◽

Glucose Levels ◽

Study Results ◽

Peptone Water

ABSTRACT The heat resistance of susceptible and multiantimicrobial-resistant Salmonella strains grown to stationary phase in glucose-free tryptic soy broth supplemented with 0.6% yeast extract (TSBYE−G; nonadapted), in regular (0.25% glucose) TSBYE, or in TSBYE−G with 1.00% added glucose (TSBYE+G; acid adapted) was determined at 55, 57, 59, and 61°C. Cultures were heated in sterile 0.1% buffered peptone water (50 μl) in heat-sealed capillary tubes immersed in a thermostatically controlled circulating-water bath. Decimal reduction times (D values) were calculated from survival curves having r 2 values of >0.90 as a means of comparing thermal tolerance among variables. D 59°C values increased (P < 0.05) from 0.50 to 0.58 to 0.66 min for TSBYE−G, TSBYE, and TSBYE+G cultures, respectively. D 61°C values of antimicrobial-susceptible Salmonella strains increased (P < 0.05) from 0.14 to 0.19 as the glucose concentration increased from 0.00 to 1.00%, respectively, while D 61°C values of multiantimicrobial-resistant Salmonella strains did not differ (P > 0.05) between TSBYE−G and TSBYE+G cultures. When averaged across glucose levels and temperatures, there were no differences (P > 0.05) between the D values of susceptible and multiantimicrobial-resistant inocula. Collectively, D values ranged from 4.23 to 5.39, 1.47 to 1.81, 0.50 to 0.66, and 0.16 to 0.20 min for Salmonella strains inactivated at 55, 57, 59, and 61°C, respectively. zD values were 1.20, 1.48, and 1.49°C for Salmonella strains grown in TSBYE+G, TSBYE, and TSBYE−G, respectively, while the corresponding activation energies of inactivation were 497, 493, and 494 kJ/mol. Study results suggested a cross-protective effect of acid adaptation on thermal inactivation but no association between antimicrobial susceptibility and the ability of salmonellae to survive heat stress.

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Degradation of keratan sulphate by β-N-acetylhexosaminidases A and B

Biochemical Journal ◽

10.1042/bj1930811 ◽

1981 ◽

Vol 193 (3) ◽

pp. 811-818 ◽

Cited By ~ 26

Author(s):

T Ludolph ◽

E Paschke ◽

J Glössl ◽

H Kresse

Keyword(s):

Isoelectric Focusing ◽

Human Liver ◽

Thermal Inactivation ◽

Sandhoff Disease ◽

Chromogenic Substrate ◽

Polymeric Substrate ◽

Keratan Sulphate ◽

Human Enzyme ◽

Tay Sachs Disease

Enzymic cleavage of beta-N-acetylglucosamine residues of keratan sulphate was studied in vitro by using substrate a [3H]glucosamine-labelled desulphated keratan sulphate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal beta-N-acetylhexosaminidases A and B are proposed to participate in the degradation of keratan sulphate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay–Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of beta-N-acetylhexosaminidase from human liver the peaks of keratan sulphate-degrading activity coincided with the activity towards p-nitrophenyl beta-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulphate-degrading activity. Both isoenzymes had the same apparent Km of 4mM, but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH–activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the p-nitrophenyl derivative.

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Thermal inactivation of tryptophan synthase. Stabilization by protein-protein interaction and protein-ligand interaction.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32629-7 ◽

1994 ◽

Vol 269 (16) ◽

pp. 11703-11706

Author(s):

S.B. Ruvinov ◽

E.W. Miles

Keyword(s):

Protein Interaction ◽

Thermal Inactivation ◽

Tryptophan Synthase ◽

Ligand Interaction ◽

Protein Protein Interaction ◽

Protein Ligand Interaction

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Methods to obtain thermal inactivation data for pathogen control in low-moisture foods

Trends in Food Science & Technology ◽

10.1016/j.tifs.2021.03.048 ◽

2021 ◽

Vol 112 ◽

pp. 174-187

Author(s):

Teng Cheng ◽

Juming Tang ◽

Ren Yang ◽

Yucen Xie ◽

Long Chen ◽

...

Keyword(s):

Thermal Inactivation ◽

Pathogen Control ◽

Low Moisture Foods

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Insight into the mechanism of thermostabilization of GH10 xylanase from Bacillus sp. strain TAR-1 by the mutation of S92 to E

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa003 ◽

2021 ◽

Vol 85 (2) ◽

pp. 386-390

Author(s):

Manami Suzuki ◽

Teisuke Takita ◽

Kohei Kuwata ◽

Kota Nakatani ◽

Tongyang Li ◽

...

Keyword(s):

Amino Acid ◽

Thermodynamic Analysis ◽

Thermal Inactivation ◽

Entropy Change ◽

Crystallographic Analysis ◽

Bacillus Sp ◽

Amino Acid Residues ◽

Gh10 Xylanase ◽

Insight Into

ABSTRACT The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95.

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