Insolubilization process increases enzyme stability

Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes

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

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented

Developing Methods to Assess Enzyme Stability in Liquid Laundry Detergents

This thesis presents work focused on developing established protein analysis methods for use in studying enzyme inactivation in laundry detergent systems. In a multi-billion dollar per year industry, basic, labour intensive procedures still dominate commercial stability studies, with extensive storage tests and activity assays remaining the industry standard. These methods are both inefficient and provide little insight into inactivation processes, leading to a 'trial and error' approach to product development. This slows the introduction new formulations and enzyme variants to the market. Furthermore, a valuable opportunity is being missed, harnessing available resources in the detergent industry to advance both protein analysis technologies and understanding of protein denaturation processes. Transfer from these basic, low throughput methods to those favoured by other protein-focused industries has been hindered by sample complexity and the presence of high concentrations of the surfactant, LAS. In this work, two novel approaches to enzyme analysis in LAS-rich media will be presented. The first employing an analogous surfactant, SDS, which yields similar effects on protein stability but does not affect UV detection, and the second, exploiting the irreversible nature of detergent enzyme unfolding to enable manipulation of formulations to within instrument specifications. These approaches will allow for incorporation of ultra-high throughput screening methods, such as DSF, as well as techniques which provide further insight into protein unfolding processes, such as CD, to the available suite of analytical techniques. Thermal data arising from this work were compared with rates of degradation obtained through conventional storage tests. Empirical fittings suggest a linear relationship between Tm values and long-term storage stability, enabling the use of thermal analysis as a tool for prediction of degradation rates. Further work is required to refine these models, however, before expanding to more complex systems

Reclamation of Marine Chitinous Materials for Chitosanase Production via Microbial Conversion by Paenibacillus macerans

[[abstract]]: Chitinous materials from marine byproducts elicit great interest among biotechnologists for their potential biomedical or agricultural applications. In this study, four kinds of marine chitinous materials (squid pens, shrimp heads, demineralized shrimp shells, and demineralized crab shells) were used to screen the best source for producing chitosanase by Paenibacillus macerans TKU029. Among them, the chitosanase activity was found to be highest in the culture using the medium containing squid pens as the sole carbon/nitrogen (C/N) source. A chitosanase which showed molecular weights at 63 kDa was isolated from P. macerans cultured on a squid pens medium. The purified TKU029 chitosanase exhibited optimum activity at 60 ◦C and pH 7, and was stable at temperatures under 50 ◦C and pH 3-8. An analysis by MALDI-TOF MS revealed that the chitosan oligosaccharides (COS) obtained from the hydrolysis of water-soluble chitosan by TKU029 crude enzyme showed various degrees of polymerization (DP), varying from 3–6. The obtained COS enhanced the growth of four lactic acid bacteria strains but exhibited no effect on the growth of E. coli. By specialized growth enhancing effects, the COS produced from hydrolyzing water soluble chitosan with TKU029 chitinolytic enzymes could have potential for use in medicine or nutraceuticals.[[sponsorship]]MOST[[notice]]補正完

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

Enzymatically Assisted CO₂ Removal from Flue-Gas

AbstractThe enzyme carbonic anhydrase is an enzyme known to enhance CO2 absorption rates. However, for economic viability in enzyme based absorption technology long term stability under process relevant conditions is needed. Thus, here enzyme stability for extended times are investigated with respect to pH, temperature and solvent. Temperatures and pH stability were tested for up to 100hours incubation and the enzyme was temperature stable up to 60°C and in the pH range from 7 to 11, with some residual activity between pH 5 and 12. Furthermore, enzyme stability was tested for 7 different capture solvents for 150 days, at 1M or 3M solvent concentrations, 40°C and pH between 8-9 and 10. Residual activity was found with all samples ranging from 12 to 91% of the initial activity. This study show that this enzyme can indeed be used for extended periods in process relevant conditions, and thus shows promise for industrial implementation as a catalyst in carbon capture

COPOLYMER HYDROGELS AS FULLY IMPLANTABLE OPTICAL BIOSENSORS: INVESTIGATING DESIGN PARADIGMS TO ACHIEVE LONG-TERM PRECLINICAL FUNCTION

Many diagnostic tests for disease management and overall health monitoring provide only an instantaneous measurement of the patient’s state of health, leaving intermediate fluctuations in biochemistry levels undisclosed. Often, fluid samples are collected periodically and analyzed using ex vivo assays. Diabetes is a prime example of this enigma where knowledge of blood biochemistry fluctuation patterns in real time could allow patients to make more informed treatment and lifestyle decisions. In recent years, hydrogels have been investigated as fully implantable biosensors by functionalizing them with enzymes and long-lifetime phosphors. However, maintaining a proper balance between enzyme stability and substrate transport when implanted has prevented preclinical proof of concept using this enzyme/phosphor sensing platform. This work explores the effect of matrix chemistry on enzyme stability and substrate transport and demonstrates the first noninvasive glucose tracking in porcine models by measuring luminescence lifetime instead of intensity. The first aim of this work focuses on poly(HEMA-co-AAm) matrices, characterizing them as glucose sensors in vitro and in vivo. A copolymer hydrogel containing 75:25 HEMA:AAm responded to up to 167 mg/dL of glucose in vitro and tracked real-time porcine blood glucose levels two hours after implantation, the first-reported real-time glucose tracking measuring phosphorescence lifetime using a noninvasive interrogation method. The second aim of this work employs alternative monomers such as dimethylacrylamide, N-vinyl pyrrolidone, and a 3- [Tris(trimethylsiloxy)silyl]propyl methacrylate to investigate enzyme stability and optimize substrate transport. These studies revealed that gels containing dimethylacrylamide and N-vinyl pyrrolidone provide the most enzyme stability, preserving between 60 and 93% of the original apparent activity after one week of incubation, but matrix inhomogeneities from adding silicone monomers can decrease sensor dynamic range by 56%. Finally, hybrid inorganic-organic interpenetrating network hydrogels were developed to prevent silicone phase separation in the hydrogels. These materials increased oxygen transport by up to 256% in vitro compared to pHEMA-based oxygen sensors and responded to modulated inspired oxygen in porcine models over 72 days. Hybrid sensors made with tissue-integrating inverted colloidal crystal architectures revealed minimal fibrosis in vivo with loosely woven collagen surrounding the implants, demonstrating promise for these hybrid materials as long-term implantable biosensors

Glucose-6-phosphate-dehydrogenase deficiency as a risk factor in proliferative disorder development

Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway (PPP) which provides reducing power (NADPH) and pentose phosphates. The former is mainly involved in the detoxification of chemical reactive species; the latter in the regulation of cell proliferation. G6PD deficiency is the most common enzymopathy in the human population, characterized by decreased G6PD activity, mainly in red blood cells, but actually also in nucleated cells. This decreased activity is not due to enzyme synthesis impairment, but rather to reduced enzyme stability, which leads to a shortening of its half-life. Therefore, a major problem is to understand the underlying mechanisms linking G6PD deficiency to oxidative stress and cell proliferation. In order to address this issue, in the present study we utilized, as an experimental model, fibroblasts isolated from pterygium, an ocular proliferative lesion, from G6PD normal and deficient (PFs+ and PFs-, respectively) patients. Our choice was determined by the fact that pterygium is believed to be caused by chronic oxidative stress induced by UV exposure, and that pterygium fibroblasts resemble a tumorigenic phenotype. As controls we utilized fibroblasts isolated from conjunctiva from G6PD normal and deficient patients (NCFs+ and NCFs-, respectively) who had undergone cataract surgery. 
Growth rate analysis revealed that PFs grow faster than NCFs, but while NCFs- grow more slowly than NCFs+, PFs- and PFs+ grow at the same rate. This was associated with significantly lower G6PD activity in NCFs+ compared to NCFs-, while no significant differences in the G6PD activity of PFs+ and PFs- were noted. This result was supported by the finding that in PFs-, G6PD mRNA levels were significantly higher than in PFs+. Another interesting finding of this study was increased green autofluorescence in both NCFs- and PFs- compared to corresponding positive cells, indicative of pronounced oxidative stress in deficient cells. Finally, abnormal accumulation of neutral lipids, mainly cholesterol esters was observed both in PFs- and PFs+ compared to NCFs- and NCFs+. Though further studies are necessary for better understanding the exact mechanism which links G6PD to oxidative stress and cell proliferation, our data allow to speculate on the role of G6PD on tumorigenesis, and to consider G6PD-deficient subjects at major risk to develop common and dreaded proliferative disorders, such as atherosclerosis and cancer. 
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Enzymatic Hydrolysis of Porang by Streptomyces Violascens BF 3.10 Mannanase for the Production of Mannooligosaccharides

Porang (Amorphophallus muelleri Blume) is an indigenous Indonesian plant containing high hemicellulose as a source of glucomannan. An alternative way to produce a good quality of mannooligosaccharides was through hydrolysis of glucomannan by endo-β mannnase from actynomicetes. Based on 16S rRNA analysis, BF 3.10 isolate, isolated from Bukit Duabelas National Park soil, Jambi was identified as Streptomyces violascens BF 3.10. Reducing sugar was analyzed by dinitrosalicylic acid methods. The highest reducing sugar was achieved at the 72 hours of incubation. Mannanase of isolate BF 3.10 had the highest activity at pH 6 and temperature of 70 °C with enzyme activity of 16.38 U/mL and was stable at 4 °C for 48 h. During 5-hour of hydrolysis with substrate concentration of 0.25%, 0.5%, and 1% porang glucomannan dissolved in 10 mL enzyme, mannooligosaccharides were produced with the degree of polymerization of 2-3. Visualization of the products by using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods showed that mannooligosaccharides produced comprised of glucose, mannobiose, mannotriose, and mannotetraose. The degree of polymerization and the simple sugars produced indicated that mannanase produced by S. violascens actively catalyzed the hydrolysis of 1.4-β-D-mannoside linkage from β-1.4-mannan backbone, that eventually produced simple sugars of mannooligosaccharides