Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.     

Thermostable and alkaline-tolerant cellulase-free xylanase produced by thermotolerant Streptomyces sp. Ab106

Cellulase-free xylanase was produced by Streptomyces sp. Ab106 using cane bagasse as the substrate at 55 degrees C. Its maximum activity was 13 IU without cellulase and mannanase activities. Its profiles were investigated. Its optimum temperature and pH were 60 degrees C and 6.0, respectively. More than 70% of its activity was remained at 60 degrees C at pH 9. This enzyme was quite stable and exhibited an active of more than 70% for 144 h at 60 degrees C, and of more than 80% for 144 h at 40 degrees C, pH 9. This thermo-tolerant and alkaline-tolerant xylanase can be used in the pulp bleaching process.     

Characterisation of potential milk coagulants from Calotropis gigantea plant parts and their hydrolytic pattern of bovine casein

The steady increase in world cheese demand keeps the search for appropriate coagulating enzymes as rennet substitutes in the scientific focus. This work reports the milk-clotting activities of aqueous crude enzyme (CE) from different parts of Calotropis gigantea using skim milk as substrate. CE from latex exhibited high caseinolytic (86.445 ± 1.055 U/ml) as well as milk-clotting activity (450 U/ml) when compared to other parts. Significant milk clotting index (MCI) was presented by crude enzyme of latex followed by stem, flower and leaf in the decreasing order (p < 0.05). CE from all parts showed an optimum activity at 37 °C, pH 5.5 and 10 mM calcium chloride concentration with excellent pH (4.5–6.5) and thermal stability (30–60 °C). Hydrolysis pattern of casein components by CE during 1 h incubation, as assessed by Tricine–SDS-PAGE, and subsequent peak analysis revealed CE from stem to be closely similar to that of rennin. However, extensive casein hydrolysis was observed when incubated with crude latex enzyme. Casein bands of CE and rennin tended to disappear as incubation progressed to 24 h. Proteolytic degradation was found to be advanced and resulted in complete breakdown of casein fractions unravelling the importance of incubation time as a possible essential requisite for controlled and appropriate casein hydrolysis. The study provides evidence of rennin-like activity associated with C. gigantea plant parts which may serve as a promising source of vegetable milk coagulant.     

The effect of fibrolytic enzymes sprayed onto forages and fed in a total mixed ratio to lactating dairy cows

We investigated the effect of spraying different combinations of fibrolytic enzymes onto forages on their nutritive value for lactating cows. Holstein cows were fed a TMR consisting of 30% corn silage, 15% alfalfa hay, and 55% concentrate (dry matter basis). During a 12-wk treatment period, the forages were treated with no enzymes (control), cellulase D and sultanas B, or cellulase D and xylanase C. Enzymes were diluted in water and sprayed onto the forages while mixing. Both combinations of enzymes supplied similar amounts of fibrolytic activity based on classical enzyme assays conducted at 50 degrees C. Cows fed forages treated with cellulase D and xylanase B tended to produce more 3.5% FCM (+2.5 kg/d) than did cows fed the untreated forages. Dry matter intake, milk production, milk fat, and milk protein were unaffected by treatment. In vitro production of gas from forages treated with enzymes was greater than from untreated forage, but 96-h volatile fatty acid production was not different among treatments. With an alternative enzyme assay based on the depolymerization of dyed substrate at 40 degrees C, activity of xylanase C was greatest at a pH of 6.5 but was substantially reduced as the pH of the assay was decreased. In contrast, xylanase B showed highest activity at pH 5 and enzyme activity was twice that of xylanase B at pH 5.5 and 6. Overall, the results of this study provide more evidence that fibrolytic enzymes can be used to improve milk production in lactating cows.     

黄粉虫浆凝乳性研究

从作用温度、酸度,Ca~(2+)浓度、底物浓度等方面,对黄粉虫浆的凝乳性进行了研究。结果表明,黄粉虫浆的最适凝乳温度为80℃;随着乳pH值的降低,黄粉虫浆的凝乳活性增高;Ca~(2+)对黄粉虫浆凝乳性有明显的促作用,随着底物浓度的增加,黄粉虫浆凝乳活性逐渐增高。并将黄粉虫浆与其他动物凝乳酶、植物凝乳酶和微生物凝乳酶进行了初步的比较。     

Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-beta-D-xyloside

Thermotoga maritima MSB8 possesses two xylanase genes, xynA and xynB. The xynB gene was isolated from the genomic DNA of T. maritima, cloned, and expressed in Escherichia coli. XynB was purified to homogeneity by heat treatment, affinity chromatography and ion-exchange column chromatography. The purified enzyme produced a single band upon SDS-PAGE corresponding to a molecular mass of 42 kDa. At 70 degrees C, the enzyme was stable between pH 5.0 and pH 11.4, and it was stable at temperatures of up to 100 degrees C from pH 7.0 to pH 8.5. At 50 degrees C, XynB displayed an optimum pH of 6.14 and at this pH the temperature for optimal enzyme activity was 90 degrees C. XynB exhibited broad substrate specificity and was highly active towards p-nitrophenyl-beta-D-xylobioside with K(m) and k(cat) values of 0.0077 mM and 5.5 s(-1), respectively, at 30 degrees C. It was also active towards p-nitrophenyl-beta-D-xyloside. The initial product of the cleavage of p-nitrophenyl-beta-D-xyloside was xylobiose, indicating that the major reaction in the cleavage was transglycosylation, not hydrolysis.     

A thermostable histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007

A thermostable histamine oxidase (EC 1.4.3.-) was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The enzyme was purified about 715-fold over the cell free extracts with a yield of 55% by ammonium sulfate fractionation and various column chromatographies. The purified enzyme was homogeneous on polyacrylamide gel-electrophoresis (native-PAGE). When the enzyme was kept at 65 degrees C and 70 degrees C for 10 min, the activity was fully stable at 65 degrees C and decreased to 9% of the initial level at 70 degrees C. The enzyme was very thermostable. The optimum pH for histamine oxidase activity was found to be at 9.0, and the enzyme was stable over the pH range of 6 to 9. The purified enzyme showed a single protein band on SDS-PAGE and its molecular mass was estimated to be about 81 kDa. The enzyme showed potent activity toward histamine, whereas it was inactive toward putrescine, cadaverine, spermine, and spermidine. Histamine oxidase was inhibited by N,N-diethyldithiocarbamate (DDTC). The inactive enzyme was restored with Cu2+ to 65% of the initial activity, but Cu+ did not enhance the enzyme activity. It is suggested that Cu2+ is essential for expression of histamine oxidase activity. The enzyme was a copper-containing protein having one atom of copper per mol of the enzyme protein as a result of atomic absorption analysis. The N-terminal amino acid sequence of the purified enzyme was different from that of histamine oxidase from Arthrobacter globiformis IFO12137.     

The proteolytic activity of the crude enzyme extracts of Baltic cod (Gadus morhua) alimentary tract

The crude enzyme extract from Baltic cod alimentary tract had maximum activity of aspartic proteinases towards haemoglobin at pH 2.0 and 3.4, and of serine proteinases with casein as the substrate at pH 8.3 and 10.4. With bovine myofibrils as the substrate the proteolysis at pH 5.0-8.0 was maximum at the lowest and highest pH values. the optimum temperature for the proteolytic activity at pH 3.4 and 10.4 was 30–45°C and at pH 8.3 it was 40–50°C. Heating the crude extract for 10 min at pH 3.4,8.3, and 10.4 in the absence of the substrate had no effect on the activity of the acid and alkaline proteinases up to about 35–40°C. the stability at higher temperature decreased gradually and total inactivation occurred at 55–60°C. In the pH range 5.5–7.5 the proteolytic activity against bovine myofibrils was low at 0°C but brought about significant loss of myosin heavy chain at 20°C.     

Purification and characterization of cis-aconitic acid decarboxylase from Aspergillus terreus TN484-M1

cis-Aconitic acid decarboxylase (CAD) was assumed to be a key enzyme in the production of itaconic acid by comparing the activity of CAD from Aspergillus terreus TN484-M1 with that of CAD from the low-itaconate yielding strain Aspergillus terreus CM85J. The constitutive CAD was purified to homogeneity from A. terreus TN484-M1 by ammonium sulfate fractionation, and column chromatography on DEAE-toyopearl, Butyl-toyopearl, and Sephacryl S200HR, and then characterized. A molecular mass of 55 kDa for the native enzyme was determined by SDS-PAGE. The enzymic activity was optimal at a pH of 6.2 and temperature of 45 degrees C. The K(m) value for cis-aconitic acid was determined as 2.45 mM (pH 6.2, 37 degrees C). The enzyme was completely inactivated by Hg+, Cu2+, Zn2+, p-chloromercuribenzoate, and 5,5'-dithio-bis(2-nitrobenzoate).     

Characterization of N-acetylmuramidase M-1 of Streptomyces globisporus produced by Escherichia coli BL21(DE3)pLysS

A gene of Streptomyces globisporus encoding N-acetylmuramidase M-1 was cloned into the pET26b vector and expressed in Escherichia coli BL21(DE3)pLysS. Maximal activity for the purified enzyme was observed at 55 degrees C with an optimal pH of 5.3, and N-bromosuccinimide strongly inhibited lytic activity even at a concentration of 0.01 mM. The enzyme showed N,O-diacetylmuramidase activity.     

Properties of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1

A NAD+-dependent medium-chain alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1 was expressed in Escherichia coli and purified. The recombinant enzyme was a homotetramer of molecular mass 1.6 x 10(2) kDa. The optimum pH for the oxidative reaction was around 10.5 and that for the reductive reaction was around 8.0. The enzyme had a broad substrate specificity including aliphatic and aromatic alcohols, aliphatic and aromatic ketones, and benzylaldehyde. This enzyme produced (S)-alcohols from the corresponding ketones. The enzyme was thermophilic and the catalytic activity increased up to 95 degrees C. It maintained 24% of the original catalytic activity after incubation for 30 min at 98 degrees C, indicating that this enzyme is highly thermostable.     

Action of rennin on kappa-casein.

The action of rennin on kappa-casein was studied as a function of time using several methods to measure activity. The first indication of rennin cleavage of kappa-casein is precipitability in .1 M acetate buffer at pH 5.2 and 5 C. A longer exposure to rennin is required to alter kappa-casein so that it forms a precipitate with calcium ions and loses its ability to stabilize alpha s-casein. The least sensitive indication of rennin activity is measurement of nitrogen soluble in 2% trichloroacetic acid. Electrophoresis experiments showed that these methods detect various stages in the conversion of kappa-casein para-kappa-casein.     

Characterization of glucoamylase immobilized on magnetic nanoparticles

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3‐aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si–O–Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 55°C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (55°C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.     

Novel antibacterial lactoferrin peptides generated by rennet digestion and autofocusing technique

Bovine lactoferrin was hydrolysed with a range of proteolytic enzymes including calf rennet, fungal rennin, and porcine pepsin. Lactoferrin hydrolysates were assessed for their antibacterial activities against Escherichia coli and Bacillus subtilis. At pH 3, calf rennet lactoferrin hydrolysate before (LFH) showed the highest antimicrobial activity, then pepsin LFH, while fungal rennin LFH was the least active. The calf rennet and pepsin LFH were fractionated using autofocusing and chromatographic techniques. The activity-guided fractionation of calf rennet LFH identified a potent antimicrobial peptide of 11-residues, lactoferricin B (Lf-cin B), and three other novel antibacterial peptides. The 11-residues Lf-cin B was the most potent antibacterial peptide and was isolated from both rennet and pepsin LFH. Pepsin LFH had a main antimicrobial peptide of 25-residues, which was not detected in calf rennet LFH. It could be concluded that calf rennet LFH had stronger antibacterial properties than porcine pepsin LFH. Besides, autofocusing could be used for scaling up the isolation of the potent rennet LFH peptides that would have a widespread commercial use as a natural food preservative substituting porcine pepsin digest, especially in Islamic communities.     

Effect of Temperatures on Fish Alkaline Protease, Protein Interaction and Texture Quality

The effect of the activity of fish alkaline protease and protein denaturation on texture changes in mullet muscle during heating were studied. The proteolytic enzyme and the urea denatured hemoglobin substrate were incubated at different pH values and various temperatures. The enzyme activity was optimal at pH 8 and at 65°C. The extent of protein-protein interactions in mullet actomyosin solutions increased with increasing temperature. The rate of protein-protein interactions increased sharply as temperature increased from 35°C to 45°C at pH 6. When mullet muscle was heated at various temperatures ranging from 55°-85°C, the shear force value increased (toughening) during the initial stage of heating and then decreased (softening). The toughening of fish flesh is probably due to muscle protein interactions, and the later tenderizing of fish flesh may be explained by alkaline protease activity (hydrolysis) masking the protein denaturation effect. When fish flesh was cooked at 100°C, no tenderizing effect was observed. This was probably due to loss of enzyme activity and only protein denaturation had significant effect on texture quality.     

PROTEOLYTIC ACTIVITY OF CRUDE ENZYME EXTRACTS OF SQUID ILLEX ARGENTINUS LIVER

The extract obtained from the acetone powder of frozen stored squid liver had optimum proteolytic activity against hemoglobin at pH 2.6 to 4.0. About 90% of the activity at pH 3.0 was inhibited by pepstatin and about 15% was inhibited by phenylmethanesulfonyl fluoride or iodoacetamide. The activity was increased by dithiothreitol and cysteine by about 55 and 40%, respectively. At pH 3 the enzyme extract had a temperature optimum of 45 to 50C; at 5C the activity was about 15% of that at 45 to 50C. At pH 7 the optimum activity was at about 45C. Heating of the extract without substrate at pH 3 and 7 caused a rapid decrease in proteolytic activity above 35 and 30C, respectively. A water extract of the liver was used to treat isolated bovine myofibrils at pH 5.5 and 0C. After 20 h a fragment of slightly lower m.w. than that of the myosin heavy chain was detected. After treatment at pH 7 and 0C, little degradation of the myofibrillar proteins was evident. At 20C the myofibrils were hydrolyzed to several products.     

Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Purification and characterization of a monoacylglycerol lipase from Pseudomonas sp. LP7315

A monoacylglycerol lipase (MGL) was purified from Pseudomonas sp. LP7315 by ammonium sulfate precipitation, anion-exchange chromatography, and preparative electrophoresis. The purified enzyme was homogeneous on SDS-PAGE with a molecular mass of 59 kDa. Its hydrolytic activity was confirmed to be specific for monoglycerides: the enzyme did not hydrolyze di- and triglycerides. MGL was found to be stable even after 1-h incubation at 65 degrees C. The optimum pH for monopalmitin hydrolysis was approximately 8. The hydrolytic activity depended not only on temperature and pH but also on the type of monoglyceride used. MGL also catalyzed monoglyceride synthesis at 65 degrees C in a solvent-free two-phase system, in which fatty acid droplets were dispersed in the glycerol phase with a low water content. The synthetic reaction proceeded at a constant rate for approximately 24 h and approximately reached an equilibrium after 48 h of reaction. The initial rate and equilibrium yield of the synthetic reaction depended on the type of fatty acid used as the substrate.     

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.