PubMed · 6596895
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1984. Enzyme engineering 7.. https://pubmed.ncbi.nlm.nih.gov/6596895/
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The present study expands previous work [(1984) Biochim. Biophys. Acta. 797, 343-347] by showing that organo-functionalized polysiloxane copolymers could entrap two of the most frequently immobilized enzymes, i.e. urease and invertase with retention of biological activity. Urease was solidly entrapped in the polymer formed from a 1:3 mixture of 3-aminopropyltriethoxysilane and tetraethylorthosilicate. The entrapment yield and the activity of the entrapped enzyme are significantly greater than with other techniques reported to date. Significantly, the entrapped enzyme possessed greater activity than its solution counterpart (36% at higher amounts of enzyme entrapped). The entrapment process also rendered the enzyme more stable toward pH and temperature, and less susceptible toward the action of urea at high concentrations. In addition, the entrapment process significantly increased the stability, both operational and storage, of the urease enzyme. When invertase was entrapped in the same copolymer, it retained two thirds of its solution activity, but the entrapment yield was lower than that of urease. Results obtained during this study also suggested that the protein may be influencing polymer development in these systems and that the resultant polymer in turn may be affecting the enzyme's activity (see following paper for further discussion).
An aqueous two-phase system of polyethylene glycol (PEG) and potassium phosphate provided a favorable environment for bioconversion of penicillin G to 6-aminopenicillanic acid (6-APA). The recombinant E. coli cells containing penicillin acylase were partitioned in the phosphate-rich bottom phase, and the product 6-APA in the PEG-rich top phase, which protected the enzyme from deactivation for a longer period of time. The continuous production of 6-APA by recycling the bottom phase showed a slight decrease in enzyme activity from initial 80 mM of 6-APA production to 61 mM during the eight days of operation at a space velocity of 0.12 hours-1. This work showed a possibility that the aqueous two-phase whole-cell enzyme bioconversion could serve as an alternative to immobilized cells on solid matrices.
A flow injection system is described for the parallel determination of pullulan and glucose during a fermentation of the fungus Aureobasidium pullulans. The polysaccharide was hydrolyzed by pullulanase and amyloglucosidase, immobilized to controlled-pore glass (CPG). The glucose produced was oxidized by glucose dehydrogenase and the NADH formed determined photometrically. The pullulan concentration was calculated from the difference to the response obtained for free glucose. The calibration curves for monomer and polymer were both linear between 2 mg dm-3 and 20 mg dm-3. Analysis of one sample for the determination of glucose and pullulan took about 10 min.