107993

23;

B. Krajewska c: ai./ Journal ojMoleruhr Caiatysis B: En^jmaiic 3 (1997)231-238

Ł

(1)

and / gives:

S₀ — $, AT_m Sq t =--r —-In —

^yjna\

changes of the above generatcd by immobiliza-tion [11].

The kinetics of inhibition of free urease has been extensivcly studied [2]. Four major classes of urease inhibitors have been investigated, namely hydroxamic acids [12-14], phospho-roamide compounds [13,15], borie and boronic acids [16,17], and heavy metal ions [18,19]. The First three classes were investigated mainly as potential therapcutie ugents against certain bac-terial urease-induced human pathogenic States, the fourtli class for analyiiea! purposes.

By contrast. studies on kinetics of inhibition of immobilized urease have scarcely been re-poned.

In this study jack bcan urease was immobilized on chitosan mernbranes. Chitosan. (1 -> 4)-2-amino-2-deoxy-/3-D-gIucan, is a deacety-latcd product of the alkali treatment of chitin which is cbtained from abundant natural rc-sources of crustaceans such as crabs. shrimps, lobsters and krills. whose shells are wastes of seafocd proccssing [20]. Chitosan. as an enzyme immobilization suppon. ołTers an attractive set of properties; it is inert, hydrophilic, biocompat-ible. it shows high affinity to proteins. and the presence of hydroxyl and amino groups facili-tates immobilization of enzymes as well as fur-ther derivatization of the polymer [21,22]. Due to its solubility in dilute organie acids. chitosan can be proccsscd into different gcometrical con-figurations: mernbranes, fibers, bollow fibers, capsules and beads [20,21,23].

In a previous report the preparation and properties of urease covalently immobilized on the chitosan membranę were described [24]. The inhibition of urease activity in native and immo-bilized forms by heavy metal ions [25] and sodium fluoride [26] were evaluated. The ure-ase-chitosan system proved to be stable and thus promising for praclical application.

In the present study the kinetics of inhibition of chitosan membrane-immobilized urease by borie acid was investigated. This inhibition is of practical signiFtcance e.g. for analysis of ureasc-based urea determinations in urinc, as borie acid is a common urine preservative used prior to analytical tests [17]. Borie acid is known to be a compctitive inhibitor for bacterial urease [16]. To estimate the reaetion kinetic constanis of the studied urease-chitosan system kinetic integration methods were used. The results are compared with those obtained for free urease [27].

2. Kinetic integration meOiods

Kinetic parameters of an enzyme-catalyzed reaetion, Michaelis constant and maximum reaetion ratę can bc obtained either by differential methods bascd on the differential Michaelis-Menten equation (Eq. (D) [28] or by integration methods based on the integrated Michaelis-Menten eąuation (Eqs. (2a) and (2b)). The former rcquirc measurements of initiai reac-tion rates at a series of substrate concentrations. whereas the latter require reeording of a single total reaetion progress curve, product conccntra-tion versus time, at ?. chosen subsirate concen-tration. Compared to differential methods, integration methods are faster. require less enz.ycne and substratc, which is why they are very useful for fast quality tests in preparing immobilized enzymes.

The Michaelis-Menten eąuation for an unin-hibited enzyme-cataiyzcd reaetion,

E + S*±ES-»E + P,

is:

dS

d7

whcrc 5 is the substrate concemration. Integrat-ing between the limits S «*= S₍, and S_t and / = 0 (2a)