The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application.

Cellobiase was coupled to a dialdehyde dextran by reductive alkylation in the presence of sodium cyanoborohydride. The resulting conjugate, obtained without loss of enzymic activity, presents properties of thermoresistance largely superior to those of native enzyme: the rate of inactivation is reduced compared to that of native enzyme and its optimal temperature of activity is 70-75 degrees C instead of 65 degrees C. Finally the conjugate presents increased longevity when subjected to experiments of operational stability; its hydrolytic activity is maintained at 60 degrees C in a 10% (w/v) cellobiose solution for more than 100 h whereas the native enzyme is inactivated after 45 h. The cellobiase-dextran conjugate was immobilized by covalent coupling on aminated silica by reductive alkylation in the presence of NaBH(3)CN. The characteristics of thermoresistance of this stabilized and immobilized conjugate were studied and compared to those of a preparation of native cellobiase immobilized on a silica support activated with glutaraldehyde. Analysis of the thermoresistance of these two cellobiase preparations clearly shows that immobilization has maintained and even enhanced their properties. In particular, the operational stability, measured at 68 degrees C on 10% (w/v) cellobiose shows an increased longevity of the stabilized and immobilized enzyme for 120 h compared to 60 h for the native immobilized enzyme. Two successive incubations of these cellobiase derivatives show that it is possible to obtain 2.5 times more glucose with the stabilized-immobilized enzyme than with the immobilized preparation. The procedure described above enables us to prepare a thermostabilized immobilized cellobiase.

The applicability of crosslinking an enzyme to an oxidized polysaccharide by reductive alkylation to enhance thermostability has been investigated for glucoamylase from Aspergillus niger. Direct covalent coupling of the enzyme to periodate-oxidized dextran in the presence of NaBH(3)CN results in a conjugate which has thermal properties similar to those of the native enzyme. Our working hypothesis postulates that enhancement of thermostability will result from rigidification of the protein's conformation subsequent to the formation of multiple covalent bonds between the protein and the support. On the basis of the known characteristics of glucoamylase from Aspergillus niger, it would seem necessary to introduce additional amino groups in the polypeptide chain of the protein. The incorporation of new amino groups was performed in two phases. First, the glycosidic part of glucoamylase was oxidized by periodate and the resulting aldehyde groups were reductively aminated by a diaminoalkane and NaBH(3)CIM. Secondly, additional amino groups were introduced on carboxyl functions into the previously aminated glucoamylase by a diaminoalkane and a water-soluble carbodiimide in the presence of maltose to protect the active site. The final derivative was then coupled to periodate-oxidized dextran T-70 in the presence of NaBH(3)CN. Starting with native glucoamylase, three successive operations give rise to a conjugate which retained 27% of the initial activity when measured with soluble starch and 39% when measured with maltopentaose. Using substrates of various sizes, it was observed that steric hindrance at the active site may result from covalent coupling to dextran T-70. It was demonstrated in heat inactivation experiments that the thermostability of the conjugate was in all cases superior to that of the native enzymes. Finally, it was observed that the operational stability of the conjugate was at least twice that of native glucoamylase at 70 degrees C on 18% maltodextrin. Additional experiments rule out the possibility that thermosta-bilization of the complex is due to other reasons than the increase in the amino content of the protein prior to crosslinking. Neither chemical modification, reticulation nor change in the net charge of the protein resulted in a derivative of glucoamylase which presented enhanced thermostability after conjugation. We conclude that for enzymes which have a low content of available amino groups, the thermostabilization method proposed previously by the present authors may still be applicable if additional amino groups are introduced into the protein prior to its crosslinking to an oxidized polysaccharide. This new example reinforces the generality of this method of stabilization.

An aldehyde derivative of riboflavin was covalently attached by reductive alkylation to soluble polycationic supports. The flavopolymers so obtained were stable under operational conditions. The catalytic efficiency towards oxidation of NADH by these flavopolymers was demonstrated, and the kinetic parameters ( K , and k,,,) revealed an overall catalytic efficiency (k,,,/K,) 185-fold greater compared to riboflavin.Various factors affecting the chemical regeneration of NAD' from NADH such as pH, ionic strength, nature of the buffer etc. were studied. The most interesting result was the highly favourable influence of borate ions which increased the reaction rate by a factor 2 -4 compared to the other buffers.The flavopolymers are very effective for in situ recycling of NAD(P)+. With up to 300-fold NADH + NAD' conversions for the system using yeast alcohol dehydrogenase and up to 1500-fold NADPH + NADP' regenerations for the system using glucose-6-phosphate dehydrogenase. These flavopolymers are superior to previous chemical recycling systems.Enzyme technology has proved its economic and industrial potential by the successful development of a 'first generation' of enzymes such as hydrolases and isomerases, which do not require coenzymes.A new objective is now to develop what are already called the 'second generation' of enzyme reactors which catalyze the synthesis and the modification of the fine chemicals of high added value. These processes are mainly oxido-reduction reactions, and thus involve the use of enzymes requiring expensive coenzymes which are needed in stoichiometry amounts. Any economically feasible process will thus depend on the retention or recovery and the regeneration of the coenzymes. Regeneration of coenzymes required for oxidoreduction reactions has been accomplished mainly by enzymatic methods [1] rather than by non-enzymatic methods.The chemical methods of regenerating coenzymes have received less attention than the enzyme-dependent processes although their advantages are stability and low cost of re-