The diffusion characteristics of several substrates of varying molecular sizes into and from Ca-alginate gel beads in well-stirred solutions were investigated. The values of the diffusion coefficient (D) of substrates such as glucose, L-tryptophan, and alpha-lactoalbumin [with molecular weight (MW) less than 2 x 10(4)] into and from the gel beads agreed with those in the water system. Their substrates could diffuse freely into and from the gel beads without disturbance by the pores in the gel beads. The diffusion of their substrates into and from the gel beads was also not disturbed by increasing the Ca-alginate concentration in the beads and the CaCl(2) concentration used in the gel preparation. In the case of higher molecular weight substances such as albumin (MW = 6.9 x 10(4)), gamma-globulin (MW = 1.54 x 10(5)) and fibrinogen (MW = 3.41 x 10(5)), the diffusion behaviors of the substrates into and from the gel beads were very different. No diffusion of their substrates into the gel beads from solutions was observed, and only albumin was partly absorbed on the surface of the gel beads. The values of D of their substrates from the gel beads into their solutions were smaller than their values in the water system, but all their substrates could diffuse from the gel beads. The diffusion of high molecular weight substrates was limited more strongly by the increase of Ca-alginate concentration in the gel beads than by the increase of the CaCl(2) concentration used in the gel preparation. Using these results, the capacity of Ca-alginate gel as a matrix of immobilization was discussed.

Screening fifteen strains of white-rot fungi for their ability to decolorize combined bleached kraft effluent showed that Coriolus versicolor in liquid culture removed over 60% of the color of the effluent within six days in the presence of sucrose. Treatment of the same effluent with this fungus, immobilized in beads of calcium alginate gel, resulted in 80% decolorization after three days in the presence of sucrose. Caustic extraction E(1) effluent was also decolorized by the immobilized fungus. Decolorization was achieved more rapidly at pH 5.0 than at pH 7.0. Recycled beads could remove color efficiently and repeatedly in the presence of air but not under anaerobic conditions.

A conical glass V-fermenter for plant cell suspension cultures is described. This V-fermenter has proved useful for the production of biomass and metabolic products. It also is ideally suited for the preparation and maintenance of actively growing cultures to be used as inocula for shake flasks and fermenters. An outline of the procedure for isolating and "conditioning" plant cell suspension cultures is presented.

Calcium alginate gel is widely and generally employed in whole microbial immobilization since its gel beads can be prepared by simple techniques under mild conditions. However, immobilization using Ca-alginate gel is inapplicable to most enzymes, since the pore size of the gel is so large that enzymes leak out from the support.' For example, a recent publication2 showed that proteins easily diffuse from the support through the pores even if their molecular weights exceed 3 X 105. It is very desirable to prevent such leakage so that Ca-alginate gel can be applied to enzyme immobilization. We have investigated whether masstransfer at the surface of the gel beads could be artificially controlled by coating the surface with polymers having a branching structure. Also, one of the authors found that Ca-alginate gel beads coated with polymers such as poly (ethyleneimine) and poly(propy1ene) had higher resistance to phosphate ions.3 In this study, we used Ca-alginate gel beads coated with poly(ethy1eneimine) (PEI) and partially quaternized polyfethyleneimine) (QPEI) to entrap glucoamylase with a molecular weight of 9.7 X 10" and found that the QPEI-coated beads have excellent features for the immobilization of the enzyme. The present communication describes a new method of entrapping glucoamylase using QPEI-coated Ca-alginate gel beads MATERIALS AND METHODSThe PEI (MW = 7 X lo4) and QPEI (degree of quaternization = 30 mol TO), obtained by treating the PEI with CH,Br, were supplied from Nihon Shokubai Kagaku Co. Ltd. (Tokyo, Japan). Sodium alginate was commercially obtained from Wako Pure Chemical Co. Ltd. (Osaka, Japan). Glucoamylase was purchased from Boehringerl Mannheim-Y amanouchi Co. Ltd. (Tokyo, Japan).The entrapment was carried out by dropping an aqueous solution containing 2% Na-alginate and 0.5% glucoamylase into a gently stirred lOOmM CaCI2 solution, and then curing the resultant gel in the solution with stirring for 2 h. These procedures were carried out in the presence of 0.5% 'enzyme to prevent the diffusion of the enzyme from the gel beads. The PEI or QPEI was coated by stirring beads (diameter = 2.8 f 0.1 mm) in the polymer solution (170, pH 5) for 5 min. The polymer-coated beads were then separated by filtration through a stainless-steel sieve and washed with enough distilled water to remove the polymer solution.The enzymatic activity of native and immobilized glucoamylase was assayed by measuring the formation rate of glucose from maltose. The native and immobilized enzymes added to the 0.2M acetate buffer solution (pH 5) containing 0.5% maltose were incubated for 30 min at 40°C with shaking. The glucose concentration was determined by the usual enzymatic m e t h~d .~ RESULTS AND DISCUSSIONThe activity loss caused by the leakage of glucoamylase out of the coated and uncoated gel beads suspended in a well-stirred, 30"C, 0.2M acetate buffer solution (50 mL, pH 5) in the glass reactor described previously2 was measured by testing the activity of the enzyme in the solution. Five hundred beads were used in e...