A new decontamination method for Bacillus subtilisin pasteurized milk: Thermosonication treatment.

Thermosonication (TS) is a novel and viable technique employed to replace conventional thermal processing. TS treatment combined with pasteurization was used to kill the residual heat-resistant Bacillus in pasteurized milk and extend the shelf life of pasteurized milk and compared with High Temperture Shoort Time (HTST) pasteurization to study its decontamination effect on Bacillus subtilis and the quality of treated milk. The results showed that after 40 kHz, 240 W, 25 min ultrasonic treatment and 50 °C heating decontamination treatment, the number of B. subtilis in the medium and milk medium decreased by 4.17 log CFU/mL and 4.09 log CFU/mL respectively. The results of cell membrane permeability showed that the leakage of DNA and protein in the HTST-TS group increased by 52.3 % and 34 %, respectively, when compared to that in the HTST group. In addition, transmission electron microscopy (TEM) analysis showed that the bacterial cell membrane of the HTST-TS group swelled up, the cell wall was ruptured, and the cell content was accumulated in the cells. The results showed that HTST-TS treatment significantly inhibited the activities of ATPase (47 %), succinate dehydrogenase (SDH) (68.6 %), and malate dehydrogenase (MDH) (54.4 %). The physical and chemical sensory evaluation of milk treated with HTST-TS showed that HTST-TS treatment could improve the L* value (2.24 %), zeta potential (64.19 %), and colloidal particle size (14.49 %) of milk but had no significant effect on oral sensitivity. In conclusion, this study provides new insights, which may be helpful in implementing this new combined decontamination method in the dairy industry to improve the quality of pasteurized milk and extend the its shelf life.

Folding and Unfolding Kinetics of Unpurified Proteins by Pulse Proteolysis.

It has been reported that pulse proteolysis may be used to investigate protein unfolding kinetics in cell lysate. However, the method has not become popular and we could not judge whether or not it is effective for protein folding study. In this work, we examined the folding and unfolding kinetics of a protein and its variants without purification by pulse proteolysis. The unfolding and refolding rates of the unpurified proteins were similar to those of the purified proteins determined by pulse proteolysis and circular dichroism. Furthermore, because we used a super-stable subtilisin as a protease, we could evaluate the kinetics at 50°C. The present work demonstrates the validity of pulse proteolysis for folding and unfolding studies of unpurified proteins.

Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: a powerful quality-control tool.

A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Šand a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Šwith an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.

Protease detection using a porous silicon based Bloch surface wave optical biosensor.

In this article we present an optical biosensor for label-free detection of trace levels of protease activity. The scheme is based on surface functionalized porous silicon optical structures which supports optical Bloch surface modes. The optical structure provides a resonant optical mode for high sensitivity detection and open access of the sensing layer to the target enzyme. Protease detection is based on the digestion of gelatin, covalently attached inside the pore space, resulting in a spectral blue-shift of the optical mode. Monitoring of spatially separated resonant optical modes is used to eliminate optical response from nonspecific adsorption.

A near real-time system for continuously monitoring airborne subtilisin-type enzymes in the industrial atmosphere.

We describe the development and validation of a portable system comprising an air sampler coupled to an automated flow injection analysis device. The system is able to monitor airborne concentrations of subtilisin-type enzymes in the workplace atmosphere on a continuous basis. Sampling is in two stages: using a sampling head that is designed to mimic human respiration at approx. 1 m s(-1) at a sampling rate of 600 l min(-1). In the second stage, the captured particles are deposited by impaction from the air stream onto the inner surface of a cyclone that is continuously washed with a jet of buffer solution. Deposited particles are then washed into a reservoir from which samples are taken every 5-6 min and injected automatically into a continuous flow injection analysis system. Proteolytic enzyme in the sample passes through a bioreactor maintained at about 40 degrees C. This contains a cellulose solid phase matrix on which is covalently immobilised Texas Red-labelled gelatin as substrate. The passing enzyme partially digests the substrate releasing fluorophore that is detected down stream in a flow cell coupled to a fluorimeter. The system is calibrated using enzyme standards and the intensity of the resulting peaks from the ex-air samples is converted to airborne concentrations using a mathematical model programmed into a PC. The system has a limit of detection of 4.8 ng m(-3) and a dynamic range of 5-60 ng m(-3). The within assay precision (RSD) is 6.3-9.6% over this range. The within batch precision is 20.3% at 20 ng m(-3) and the corresponding between batch value is 19.5%. The system has been run for periods up to 8 h in the laboratory and for up to 4 h at a factory site and the values obtained compared with time-averaged values obtained from a conventional Galley sampler and in-house analysis when reasonable agreement of the results was observed. The stability of the system over 21 days of continuous use with standards injected periodically was studied. Linearity was observed for all the standard plots throughout. At the end of 21 days, after a total exposure equivalent to 2395 ng ml(-1) of Savinase, the signal due to the 5.0 ng ml(-1) standard was still easily detectable.

Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae)

Con el objetivo de caracterizar aislamientos para el control biológico demosquita blanca (Homoptera: Aleyrodidae), se estudiaron tres cultivosmonospóricos (CMs) de Paecilomyces fumosoroseus, hongo entomopatógenode la mosquita blanca (Homoptera: Aleyrodidae). Se determinó la virulencia enninfas de segundo estadio de la mosquita blanca expresada comoconcentración letal media (CL50). Se determinó la producción de propágulosfúngicos (blastosporas, cuerpos hifales, hifas cortas, conidios sumergidos)en medio líquido, se midió la relación de largo y ancho de los propágulos,y la actividad proteolítica (total; tipo subtilisina, Pr1 y tipo tripsina, Pr2)simultáneamente con la producción de propágulos. La proteasa total sedeterminó con azocaseina, y las actividades de Pr1 y Pr2 con N-Succinil-Ala-Ala-Pro-Phe-p-nitroanilida y N-Benzoil-Phe-Val-Arg-p-nitroanilida comosustratos específicos, respectivamente. Se observó variabilidad entre los tresCMs en cuanto a la virulencia, producción de propágulos y proteasas. Losbioensayos mostraron una CL50 de 1,1 x 103, 2,5 x 104 y 7,6 x 104 conidios/mlpara los CMs EH-506/3, EH-503/3 y EH-520/3, respectivamente.El CM donde se observó el mayor número de propágulos en las condicionesensayadas fue EH-503/3 (7,7 x 107), luego EH-520/3 con 6,4 x 107 y EH-506/3con 1,0 x 107 propágulos/ml. Las actividades enzimáticas de Pr1 y Pr2 sedemostraron en los tres CMs. La actividad tipo subtilisina (Pr1) fue mayor enel aislado más virulento (EH-506/3) con 745,7 UPr1/ml a las 120 h, y señala aPr1 como un marcador fenotípico de virulencia para P. fumosoroseus.EH-506/3 es un buen candidato para el control biológico de la mosquitablanca en México, por su alta virulencia, elevada concentración de Pr1 y surápida transición a blastosporas en el medio líquido ensayado(AU)

To properly characterize several isolates of Paecilomyces fumosoroseus,a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and otherinsect pests for biocontrol purposes, virulence towards Trialeurodesvaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activityduring propagule production were investigated in monospore cultures (MCs).The virulence of three MCs towards second instar whiteflies was measuredand expressed as lethal median concentration (LC50). Number andwidth–length ratio of propagules (blastospores, hyphal bodies, short hyphae,submerged conidia) and extracellular proteolytic activity was determinedsimultaneously in liquid medium. Total protease activity was assayed withazocasein, Pr1 and Pr2 activity was determined with the substratesN-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide,respectively. Natural variability in virulence, propagule productionand cuticle-degrading proteases among isolates was observed. Bioassaysshowed a LC50 of 1.1 x 103, 2.5 x 104 and 7.6 x 104 conidia/ml for MCsEH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the mostvirulent isolate. Isolate EH-503/3 produced the highest yield of propagules(7.7 x 107 propagules/ml), followed by EH-520/3 with 6.4 x 107 and EH-506/3with 1.0 x 107 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activitywas present in the three MCs. Subtilisin-like (Pr1) activity was highest(745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears tobe a good candidate for whitefly biocontrol due to its high virulence,Pr1 concentration and rapid transition to blastospores in submerged liquidmedium(AU)

[Exoproteinases of the oomycete Phytophthora infestans]. / Ekzoproteinazy oomitseta Phytophthora infestans.

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown by SDS-PAGE with the presence of gelatin to include at least six components differing in molecular weight. Inhibitory analysis and study of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases specific to trypsin and subtilisin and metalloproteinases. Their activity is suppressed by proteinase-inhibitor proteins from potato tubers. It is suggested that P. infestans exoproteinases may be the metabolic target for natural proteinase inhibitors from potato.

[Membrane-bound forms of serine proteases of Bacillus intermedius]. / Membranno-sviazannye formy serinovykh proteinaz Bacillus intermedius.

Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes.

[Synthesis and secretion of Bacillus intermedius proteinases in the late stages of sporulation]. / Sintez i sekretsiia proteinaz Bacillus intermedius na pozdnikh stadiiakh sporoobrazovaniia.

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating beta-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.

Sol-gel immobilization of serine proteases for application in organic solvents.

The serine proteases alpha-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of alpha-chymotrypsin and the resulting specific enzyme activity in the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol in cyclohexane is 43 times higher than that of a nonimmobilized lyophilized alpha-chymotrypsin. The activities of trypsin and subtilisin Carlsberg are enhanced with 437 and 31 times, respectively. The effect of immobilization on the enzyme activity is highest in hydrophobic solvents.