Retention enzyme activity

In accordance to the enzyme stability data towards pH and temperature the UF experiments for LiP concentration were performed at pH 7.0 and controlled temperature of 25 °C. The best results were obtained using a PS 10 membrane that showed 96% of enzyme activity retention in the concentrate. The CA20 membrane showed a lower decrease in total permeability (Lp) in comparison to the PS 10 membrane suggesting a relative higher LiP adsorption and pore blocking. 

A major concern in spray drying of enzymes is the retention of their activities, whereas this complication is not seen in the case of purely chemical systems. Therefore, the enzyme activity retention must be close to 100% in the spraydrying operation and moreover, the shelf life of the dried enzyme products must be excellent, i.e., enzyme activity must be retained for long-time storage. 

Use of ultrafiltration (UF) membranes is becoming increasingly popular for clarification of apple juice. AH particulate matter and cloud is removed, but enzymes pass through the membrane as part of the clarified juice. Thus pasteurization before UF treatment to inactivate enzymes prevents haze formation from enzymatic activity. Retention of flavor volatiles is lower than that using a rack-and-frame press, but higher than that using rotary vacuum precoat-filtration (21). 

Figure 5.12 Hydrolysis of methylparathion to p-nitrophenol. The cross-linked phospholipid-based nanocapsules are permeable to reactant and product, while allowing for the retention of enzyme activity. Reproduced with permission from [92].

Extracellular enzymes are rapidly sorbed at mineral and humic colloids in soils and sediments. Mineral colloids have a high affinity for enzymes although that is not always synonymous with the retention of their catalytic ability. On the other hand, humic substances have the ability to sorb and sequester enzymes in such a way as to retain their catalytic activity they could also strongly inactivate enzyme activity depending on interaction mechanisms. 

The decolorization of Orange II by immobilized P. chrysosporium in a continuous packed-bed reactor (PBR) was investigated [50]. Nearly complete decolorization (95%) with immobilized fungus on PuF was obtained when working at optimal conditions [dye load rate of 0.2 g/l/d, temperature of 37°C, a hydraulic retention time (HRT) of 24 h], and also oxygen gas in a pulsed flow was applied. A correlation between residual MnP activity and decolorization was observed, but no laccase and LiP enzyme activities were detected. 

Statistical Methods. Means of treatment groups for plasma retention of BSP, plasma osmolality, total plasma protein concentration and urine flow rates were compared by students t test for independent sample means (17). Plasma enzyme activity data were converted to a quantal form and analyzed by the Fischer Exact Probability Test (18). Values greater than 2 standard deviations (P < 0.05) from the control value were chosen to indicate a positive response in treated fish. 

The retention of 1,2-dibromoethane in tissues and body fluids can be altered by concurrent exposure to modifiers of enzyme activity, such as disulfiram (Plotnick et al. 1979). The concentration of radiolabeled 1,2-dibromoethane in the liver, kidneys, spleen, testes, and brain increased significantly in rats fed disulfiram in the diet for 12 days before an oral dose of 15 mg C-1,2- dibromoethane/kg compared with rats not fed disulfiram. Disulfiram, an inhibitor of P-450 metabolism (via action on acetaldehyde dehydrogenase), was found to increase the uptake of C into liver nuclei. These observations correlate well with the results of chronic studies (Wong et al. 1982) that demonstrated enhanced tumorigenic effects in the liver and testes following combined 1,2-dibromoethane and disulfiram exposure. 

Figure 2. Comparisons of cellulose degradation of the MSW feedstock with specific cellulase enzyme activities in sludge from 7 CSTR digesters operated under different retention times and various conditions of nutrient limitation.

Figure 5. HPLC profiles of products from reactions catalyzed by secreted pectate lyase activities from Erwinia chrysanthemi after 66 min and 192 min. Reaction mixtures contained 4 units of enzyme activity per ml and 0.1% PGA in 0.05 M Tris-HCl, pH 8.5, 0.2 mM CaClg. Injections of 0.05 ml were made from reaction mixtures with a WISP automatic sample injector and eluted at 2.0 ml/min with a run time of 60 min. Compounds eluting with retention times of 5.36, 6.40, 7.76, and 9.56 min corresponded to unsaturated oligogalacturonate reference standards with DP values of 2, 3, 4, and 5, respectively.

Wehtje, E., Adlercreutz, P. and Mattiasson, B. (1993) Improved activity retention of enzymes deposited on solid supports. Biotechnol. Bioeng., 41, 171-178. 

Amylases. In the case of pome fruits other enzyme activities are sometimes required. When fruit has been picked before maturity and then ripened under controlled atmospheric conditions in a cool store, there is a likelihood of starch retention originating from the unripe fruit. This starch can become gelatinised during juice processing and can give rise to precipitation and haze effects in the final product. Amylases are used here to break down any residual starch and overcome such problems. 

The versatility of water-soluble polyphosphazenes is in the variations in the structures that can be prepared. Structures with a low glass-transition temperature backbone can be modified with a variety of versatile side units. These may find use in solid polymeric ionic conductors, as a means to entrap and immobilize enzymes with retention of enzymic activity, and in biological functions as hydrogels with the capability of exhibiting biocompatibility and... 

Rat brain microsome preparations were conveniently stored at -10C with retention of enzyme activity. Solubilization with Triton X-100 appears to be effective and the solubilized enzyme preparation, after filtration once with Amicon XM-300 diaflo membrane, was introduced into an isoelectric focusing column (LKB) with an ampholine pH range of 3.5-10. 

The enzyme, normally in solution, is added and after aging either a thin film or a gel can be formed with the encapsulated molecules. Depending upon the acid used, the solution pH and other conditions, gelation can take from 1 min up to several days. The sol-gel matrix shows many advantages (i) the ability to entrap a large amount of enzyme, (ii) retention of the enzymatic activity due to the sufficiently mild conditions of the sol-gel process,... 

Sensor fabrication occurs in two steps. The first step is the immobilization of GOx on the surface of the nanotube. This is accomplished by adding GOx to a solution of surfactant stabilized nanotubes and dialyzing away the surfactant. Dialysis is an ideal method for assembling enzymes on a nanotube surface, because the method allows retention of enzyme activity while simultaneously maintaining nanotube colloidal stability. The resulting GOx-S WNT solution exhibits a shift in the nanotube fluorescence indicative of the enzyme layer being less tightly packed around the nanotube than the surfactant layer. The second step is addition of ferricyanide to the GOx-SWNT solution. Adsorption of ferricyanide to the nanotube surface... 

Polyelectrolyte multilayer microspheres, prepared by alternating adsorption of dextran sulfate and protamine on melamine formaldehyde cores followed by the partial decomposition of the core, were used to immobilise the peroxidase and glucose oxidase. Retention of enzymic activity of the peroxidase/glucose oxidase system incorporated into the microspheres was demonstrated. These bienzyme system immobilised in the microspheres can be applied for kinetic glucose assays [ 156]. 

Elcd enzyme showed better survival rates with individual heat or ammonia treatment (up to 67 and 49%, respectively), but the combination of heat and ammonia in AFEX treatment caused a drastic loss in the activity of Elcd. The maximum observed activity retention in AFEX-treated transgenic tobacco plants was only 35% at 60°C, 0.5 1 ammonia loading ratio, and 40% moisture content. Future studies may be able to clarify the mechanistic bases for these observations. 

The addition of polyhydroxyl compounds to enzyme solutions have been shown to increase the stabilities of enzymes, (13,16,19,20). This is thought to be due to the interaction of the polyhydroxyl compound, (e.g. sucrose, polyethylene glycols, sugar alcohols, etc), with water in the system. This effectively reduces the protein - water interactions as the polyhydroxy compounds become preferentially hydrated and thus die hydrophobic interactions of the protein structure are effectively strengthened. This leads to an increased resistance to thermal denaturadon of the protein structure, and in the case of enzymes, an increase in the stability of the enzyme, shown by retention of enzymic activity at temperatures at which unmodified aqueous enzyme solutions are deactivated. 

---