Enzymes sarcosine oxidase

In this section, we summarize information about five Ci enzymes involved in metabolism of 5-formyl-THF (5-CHO-THF) and formate, whose existence was predicted from genomics data. 5-Formyltetrahydrofolate cycloligase (5-FCL) and S-formylglutathione hydrolase have since been shown to catalyze the anticipated reactions the other three enzymes (10-formyl-THF deformylase, formamidase, and sarcosine oxidase) are still putative. 

CHIKAYAMA, M., OHSUMI, M., YOKOTA, S., Enzyme cytochemical localization of sarcosine oxidase activity in the liver and kidney of several mammals, Histochem. Cell Biol., 2000,113,489-495. 

In an early application, an enzyme electrode system was reported for the determination of creatinine and creatine, using a combination of creatinine amidohy-drolase, creatine amidinohydrolase and sarcosine oxidase, co-immobilized on an asymmetric cellulose acetate membrane. Thus, the hydrogen peroxide produced was detected to give a quantitative measure of creatine and creatinine in biological fluids [70]. 

An interesting application for the oxidation of organic compounds is of electrochemical nature. Octacyano complexes have been used to monitor redox enzymes such as lactate oxidase (from Pediococcus sp.) and sarcosine oxidase (from Arthrobactersp.) in a suitable electrochemical system (114). Two equivalents of [M(CN)g] can, for example, be oxidized at the electrode surface to [M(CN)g], which in turn can oxidize the flavoproteien to its oxidized form. This in turn reacts with, for example, L-lactic acid to produce pyruvic acid. 

The preferred absorption-based methods are those based on enzyme-catalyzed reactions. The absorbance of NADH (340 nm) after addition of creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, and NAD to a creatine-containing solution is proportional to the concentration of creatine present. Creatinine amidohydrolase produces creatine from creatinine. Thus, any assays for creatinine which use creatinine amidohydrolase in the first step can be used for creatine by just leaving out that first step. Conversely, creatine can interfere in those analyses, although creatine is normally present in substantially lower concentrations than creatinine. 

When the three-enzyme sequence based on creatinine amidohydrolase is used, any creatine present can interfere with the determination of creatinine, so two sensors are used one to determine the total creatine plus creatinine and one to determine just creatine (by only using creatine amidinohydrolase and sarcosine oxidase). Creatinine is determined by difference. Amperometric sensors are generally based on this sequence and do not suffer from interferences. They are usually designed to respond to peroxide, though some have used oxygen electrodes. Typically, Pt electrodes are used. A sensor for just creatine only requires the creatine amidinohydrolase and sarcosine oxidase sequence. 

A three-enzyme electrode system, such as needed for creatinine measurement, poses a more difficult enzyme-immobilisation problem, in that different enzymes have different immobilisation requirements and their microenvironmental interrelationships need to be optimised. For one creatine sensor, the requisite creatine amidinohydrolase and sarcosine oxidase were immobihsed in polyurethane pre-polymer and PEG-hnked creatinine amidohydrolase was attached via diisocyanate pre-polymer to create a polyurethane adduct [14]. The likelihood of enzyme inactivation with chemical immobih-sation is high, but provided an enzyme preparation survives this, long-term stability is feasible. In the case of these three particular enzymes, a loss of activity resulted from silver ions diffusing from the reference electrode the material solution was to protect the enzyme layer with a diffusion-resisting cellulose acetate membrane. 

Figure 3.1 Interference removing oxidising membrane based on incorporated Pb02 particles over a three-enzyme creatinine biosensor based on creatinine amidohydrolase (CA), creatine amidinohydrolase (Cl) and sarcosine oxidase (SA).

From the different cases related to inhibition, the most in-depth studied case is that of pesticides, mainly as inhibitors of cholinesterase enzymes other cases involve inhibition of sarcosine oxidase that allows determining carboxylic acids or inhibition of protein phosphatases, attempted for the resolution of microcystin types. Inhibition of aminooxidases also opens a way for determination of amount and effects of modem antidepressants. 

The coreticulation described above [3] is simple to perform during multienzymatic immobilization. The required enzymes are mixed with albumin, and enough glutaraldehyde is added to cross-link the various proteins. This method is also used for the coimmobilization of creatinin amidohydrolase, creatin amidinohydrolase and sarcosine oxidase, to construct a creatinin electrode, and for the coimmobilization of a-glucosidase and glucose oxidase for the determination of starch [40]. 

Most studies have focused on amperometric glucose sensors based on GOx or GOx/ peroxidase [168-182]. However, a wide variety of other enzymes, including peroxidase, tyrosinase, choline oxidase, sarcosine oxidase, cholesterol oxidase, xanthine oxidase, glutamate oxidase, glutamate dehydrogenase, nitrate reductase, cholinesterase, cholesterol esterase, uricase, urease, creatinase, and creatininase, have also been employed for bioelectrosensor construction [183-203]. Representative examples are outlined in Table 2. 

---