Properties of Endoglucanase of Penicillium chrysogemum PCL501

Crude extracellular enzyme from a 3-day culture of Penicillium chrysogenum (PCL 501), in basal medium containing cellulose as the sole carbon source, yielded 0.67 ± 0.03, 19.94 ± 1.30 and 8.50 ± 0.50 units mg protein-1 of 1, 4- â-endoglucanase, â-glucosidase and xylanase activity respectively. The crude enzyme was subjected to ammonium sulphate precipitation (80% saturation) and gel filtration. A purification-fold of 7.5 was achieved. Two active fractions of 1, 4 âendoglucanase (EC 3. 2. 1. 4), which exhibited about the same activity towards carboxymethylcellulose (CMC), were obtained and pooled for the subsequent analyses. The endoglucanase gave a Vmax of 10.0 ± 0.4 μmol min-1 mg protein-1 and Km of 11.8 ± 0.4 gL-1 with CMC. The enzyme was most active at pH of 4.5 – 5.0 and temperature range of 40 – 50 OC. The optimum pH was 4.9 while the Optimum temperature was 48 OC. Divalent metal ions and EDTA affected the enzyme activity at 2.0 mM concentrations. Mn2+ and Fe2+ had stimulatory effects on the enzyme whereas Mg2+, Cu2+, Zn2+, Hg2+ and EDTA inhibited the enzyme activity. The effect of Ca2+ was not significant. Over 3- fold increase in the enzyme activity was recorded with Mn2+. Percentage inhibition of 65.9 and 79.7 respectively was obtained with Hg2+ and EDTA. The organism appears to produce two types of endoglucanase which differed in their molecular weight but not significantly in their activity. The enzyme activity was highly stimulated by manganese ion and inhibited by the metal-chelating agent, EDTA

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity–potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity–potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity–potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies

A new intrinsic thermal parameter for enzymes reveals true temperature optima

Two established thermal properties of enzymes are the Arrhenius activation energy and thermal stability. Arising from anomalies found in the variation of enzyme activity with temperature, a comparison has been made of experimental data for the activity and stability properties of five different enzymes with theoretical models. The results provide evidence for a new and fundamental third thermal parameter of enzymes, Teq, arising from a subsecond timescale-reversible temperature-dependent equilibrium between the active enzyme and an inactive (or less active) form. Thus, at temperatures above its optimum, the decrease in enzyme activity arising from the temperature-dependent shift in this equilibrium is up to two orders of magnitude greater than what occurs through thermal denaturation. This parameter has important implications for our understanding of the connection between catalytic activity and thermostability and of the effect of temperature on enzyme reactions within the cell. Unlike the Arrhenius activation energy, which is unaffected by the source (“evolved”) temperature of the enzyme, and enzyme stability, which is not necessarily related to activity, Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological, and environmental aspects of the adaptation of life to temperature in a way that has not been described previously. We may therefore expect the effect of evolution on Teq with respect to enzyme temperature/activity effects to be more important than on thermal stability. Teq is also an important parameter to consider when engineering enzymes to modify their thermal properties by both rational design and by directed enzyme evolution

Kinetic Study and Characterization of 1,4-β-Endoglucanase of Aspergillus niger ANL301

Submerged fermentation of Aspergillus niger ANL 301 in basal medium containing cellulose as sole carbon source, yielded crude extracellular proteins with 0.54 ± 0.02 units mg protein-1 of 1,4-β-endoglucanase activity. Partial purification by ammonium sulphate precipitation (80% saturation) and gel filtration on Sephadex 25-300 gave two active fractions of 1,4-β-endoglucanase, which exhibited close activity towards carboxymethyl-cellulose (CMC). The pH profile of the pooled enzyme fractions showed three activity peaks at pH 3.5, 5.5 and 7.0. The enzyme was most active at pH 5.5 and showed optimal activity at 50°C. Vmax of 4.4 ± 0.4 µmol min-1 mg protein-1 and Km of 12.5 ± 0.4 gL-1 was obtained with CMC for the enzyme. Different divalent metal ions and EDTA affected the enzyme activity at 2.0 mM concentrations in different ways. Mn2+ and Fe2+ exhibited 253.4 and 24.0% stimulatory effects, respectively on the enzyme activity. Mg2+, Ca2+, Cu2+, and Zn2+ inhibited the enzyme by between 22.3 and 29.4%, whereas 75.0 and 71.3% inhibition were obtained with Hg2+ and EDTA, respectively. Manganese ion showed an exceptional activation of the 1,4-β-endoglucanase. The organism produced two types of 1,4-β-endoglucanase with different molecular weights

Effect of the ionic liquid [bmim]Cl and high pressure on the activity of cellulase

The effect of the ionic liquid 1-butyl-3-methylimidazolium chloride ([bmim]Cl) and of high pressure on the activity of cellulase from Aspergillus niger were studied separately and in combination. The enzyme activity decreased with increasing concentrations of [bmim]Cl, reaching 50% the value in aqueous buffer with 20% [bmim]Cl. However, when the enzyme is held in 10% [bmim]Cl and is then assayed in 1% [bmim]Cl, it showed only 8% reduction of activity. These results can be explained by the fact that the activity of the enzyme in [bmim]Cl is linearly correlated with the decrease of the thermodynamic water activity (aw). Under pressure the enzyme activity varied from less 60% (at 200MPa) to equal (at 400 MPa), compared to atmospheric pressure. In 10% [bmim]Cl under pressure, cellulase activity is improved compared to atmospheric pressure, varying from equal (at 600 MPa) to 1.7-fold higher (at 100 MPa). This opens the possibility to improve cellulase activity in ionic liquids, and possibly of other enzymes, by carrying out the reaction under pressure

Paraoxonase 1 Polymorphism p.Q192R in Patients With Dementia

PON1 PCR-RFLP polymorphism frequency and enzyme activity were determined in 223 patients with dementia (94 with AD, 55 with VaD and 74 with MD) and in 100 age and sex matched controls without dementia. I found no statistical significance of genotype frequencies between analyzed groups. 
Paraoxonase 1 activity was lower in patients carrying the R allele in locus 192 as compared with the R allele non-carriers. The results showed statistically significant association between PON1 polymorphism and enzyme activity and thus can suggest an important relationship between different isoforms of PON1, enzyme activity and dementia

Astroenzymology – the environmental limits of enzyme activity

Using organisms from extreme terrestrial environments as models for extraterrestrial life may lead us to underestimate the range of environments that life may inhabit. An alternative approach is to inspect the range of conditions over which crucial biomolecules might function. Recent investigations of enzyme activity suggest that they have the potential to function over a wider range of environmental conditions than expected. Although the upper temperature limit for enzyme stability is unclear, some enzymes are active up to 130°C. The evidence is that the instability of enzymes is a functional requirement, rather then because of any restraint on achieving higher stability. There is no evidence that enzyme activity ceases at low temperatures; it declines in a predictable manner to the lowest temperature at which it has been possible to make measurements, -100°C. It has been generally accepted that dehydration stops enzyme activity but this acceptance may have arisen partly from the technical difficulty of assessing enzyme activity without a fluid medium for diffusion. Experiments using anhydrous organic solvents or gas phase substrates suggest activity occurs in enzymes at very low hydration

Cellulase Production by Wild-type Aspergillus niger, Penicillium chrysogenum and Trichoderma harzianum Using Waste Cellulosic Materials

Waste cellulosic materials (corncob, sawdust and sugarcane pulp) and crystalline cellulose induced cellulase production in wild strains of Aspergillus niger, Penicillium chrysogenum and Trichoderma harzianum isolated from a wood-waste dump in Lagos, Nigeria. Cellulose-supplemented media gave the maximum cellulase activity of 0.54, 0.67 and 0.39 units mg Protein-1 for A. niger, P. chrysogenum and T. harzianum respectively. The maximum enzyme activity for A. niger was obtained at 36 h of cultivation, while P. chrysogenum and T. harzianum gave their maximum enzyme activities at 12 and 60 h respectively. For the cellulosic wastes, highest enzyme activity was obtained with sawdust where A. niger, P. chrysogenum and T. harzianum gave the maximum enzyme activity of 0.30, 0.24 and 0.20 units mg Protein-1 respectively after 144 h of cultivation. A. niger recorded the highest enzyme activity with any of the three cellulosic materials followed by P. chrysogenum. It thus appears that the use of sawdust presents the best option for low-cost commercial production of cellulase using A. niger and P. chrysogenum as discussed herewith

Actinidin : the predominant protease in kiwifruit : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Food technology at Massey University, Manawatū, New Zealand

Kiwifruit protein (actinidin) has been widely known as a protease. Kiwifruit protein has the potential of utilization in food industry as an enzyme that aids food digestion. In this project, the soluble kiwifruit proteins were extracted from fresh Hayward and SunGold kiwifruit. Soluble kiwifruit proteins were analysed by the Hartree-Lowry method, SDS-PAGE, enzyme activity determination, ion-exchange chromatography and mass spectrometry. Anti-actinidin antibodies were raised by the injection of purified actinidin into rabbits. The main soluble kiwifruit protein was recognized by anti-actinidin antibodies using Western blot. Moreover, the effects of post-harvest storage on protein content, total enzyme activity and specific enzyme activity were investigated. Comparable studies on both Hayward and SunGold kiwifruit were also carried out in this project. The results showed that Hayward and SunGold kiwifruit had a similar protein content. However, the total enzyme activity of Hayward kiwifruit was about 8 times higher than that of SunGold kiwifruit. The protein with enzyme activity (active actinidin) had a molecular weight of about 27 kDa according to SDS-PAGE and was one of main soluble proteins in Hayward and SunGold kiwifruit. This protease was purified from fresh kiwifruit by anion-exchange chromatography. A polyclonal antibody against actinidin was successfully generated in a rabbit using purified actinidin. Protein with a molecular weight of 27 kDa was recognized by the anti-actinidin antibodies. Post-harvest storage at 1 °C for up to 12 weeks significantly increased the total and specific enzyme activities of SunGold kiwifruit (P<0.05). By contrast, the total and specific enzyme activities of Hayward kiwifruit had a significant decrease after 16 weeks’ storage (P<0.05). Hayward kiwifruit had no significant changes in protein content after storage (P<0.05) while the protein content of SunGold kiwifruit fluctuated in a range from 5.04 to 5.84 mg/mL during post-harvest storage. This study may help to understand the nature of kiwifruit proteins with enzyme activity, which contributes to a full understanding of the health benefits of kiwifruit

Assaying activity and assessing thermostability of hyperthermophilic enzymes

There is now a wide variety of intra- and extra-cellular enzymes available from organisms growing above 75°C, and having sufficient stability to allow assay well above this temperature. For some of these enzymes, to assay below even 95°C will involve measurement below the optimal growth temperature for the organism. The purpose of this chapter is to cover practical aspects of enzyme assay procedures that are specific to high temperatures. Since by far the commonest routine assessment of enzyme stability is activity loss, and because it is always unwise to measure enzyme activity without being confident of its stability during the assay, we include an outline of procedures for measuring enzyme activity loss/stability at high temperatures