The ability to photoregulate enzyme activities could provide important new opportunities for development of diagnostic assays, sequential bioprocessing, and lab assays in both traditional and microfluidic formats. We show here that the photoinduced changes in the size and hydration of a ''smart'' polymer chain coil can be used to regulate substrate access and enzyme activity when conjugated to the enzyme at a specific point just outside the active site. The photoresponsive polymers thus serve jointly as antennae and actuators that reversibly respond to distinct optical signals to switch the polymer-enzyme conjugates on and off, and work when the conjugate is free in solution or when immobilized on magnetic beads. smart polymer ͉ photoswitch ͉ biotechnology ͉ bioMEMs ͉ bioconjugates

A method for thermally induced switching of enzyme activity has been developed, based on the site-directed conjugation of end-reactive temperature-responsive polymers to a unique cysteine (Cys) residue positioned near the enzyme active site. The reversible temperature-induced collapse of N,N-dimethylacrylamide (DMA)/N-4-phenylazo-phenylacrylamide (AZAAm) copolymers (DMAAm) has been used as a molecular switch to control the catalytic activity of endoglucanase 12A (EG 12A). The polymer was conjugated to the EG 12A site-directed mutant N55C, directly adjacent to the cellulose binding cleft, and to the S25C mutant, where the conjugation site is more distant. The N55C conjugate displayed a larger activity shutoff efficiency in the collapsed polymer state than the S25C conjugate. Increasing the polymer molecular weight was also shown to increase the shutoff efficiency of the switch. Related to these effects of conjugation site and polymer size, the switching efficiency was found to be strongly dependent on substrate size. With a small substrate, o-nitrophenyl-beta-d-cellobioside (ONPC), there was minimal blocking of enzyme activity when the polymer was in the expanded state. With a large substrate, hydroxyethyl cellulose (HEC), there was a large reduction of enzyme activity in the polymer expanded state, even with relatively small polymer chains, and a further reduction when the polymer was collapsed. Similar general trends for the interactive effects of conjugation site, polymer size, and substrate size were observed for immobilized conjugates. Kinetic studies demonstrated that the switching activity was due to the blocking of substrate association by the collapsed polymers. These investigations provide mechanistic insight that can be utilized to design molecular switches for a variety of stimuli-responsive polymer-protein conjugates.

We have cloned and determined the nucleotide sequence of the gene encoding an extracellular beta-glucosidase (bgl1) from the cellulolytic fungus Trichoderma reesei. The predicted open reading frame of the bgl1 gene is interrupted by two putative introns of 70 and 64 bp and encodes a protein with a calculated molecular weight of 75,341. The genomic segment encoding bgl1 was cloned into a vector that contained the selectable marker gene, amdS. Transformation of T. reesei with this vector resulted in several stable transformant strains all possessing an increased copy number of the bgl1 gene integrated into the genome together with elevated rates of glucose production from avicel. One transformant produced an extracellular cellulase with a five-fold increase in the rate of production of glucose from cellobiose, a 33% rate increase from avicel, and a 17% increase from phosphoric acid swollen cellulose. These data suggest that the cellulolytic activity of T. reesei strains may be specifically improved by transformation with cloned cellulase genes.

We have used a targeted gene deletion event to remove the coding region for the bgl1 gene encoding an extracellular beta-glucosidase from the genome of the cellulolytic fungus Trichoderma reesei. The bgl1 null mutants were used to investigate the role of beta-glucosidase in the hydrolysis of cellulose and induction of the other cellulolytic enzyme components. In the absence of extracellular beta-glucosidase, growth of bgl1 null strains on several carbon sources was the same as that of the parent (as measured by mycelial dry weight). However, levels of extracellular protein and total endoglucanase production were seen to lag relative to those levels observed in the control strain. The mRNA levels of the CBHI, CBHII, EGI, and EGII cellulase genes (cbh1, cbh2, egl1 and egl3) showed a corresponding lag in induction, suggesting that the absence of extracellular beta-glucosidase has an effect on the co-ordinate regulation of the other cellulase genes at the level of transcription. The addition of a potent inducer of the cellulase complex (sophorose) resulted in normal rates of cellulase gene mRNA production and extracellular protein release. This indicates that the absence of beta-glucosidase is not affecting some intrinsic cellular ability to produce mRNA or secrete protein. These data suggest that a functional beta-glucosidase is at least partially responsible for the efficient induction of the depolymerase enzymes of the cellulase complex. The observation that the cellulase complex is induced, albeit after a lag, suggests that other enzymes are present that can substitute for the function of beta-glucosidase during induction.

The role of abscisic acid (ABA) in the signal transduction pathway associated with NaCl-induced up-regulation of antioxidant enzyme activity was examined in a NaCl-tolerant cotton callus cell line treated with NaCl, ABA, paraquat, or H2O2 in the presence and absence or fluridone, an inhibitor of terpene, and therefore, ABA synthesis. Treatment with NaCl resulted in a rapid increase (within 30 minutes) in the ABA levels of the callus tissue, and the NaCl, ABA, and paraquat treatments induced rapid increases in the activities of superoxide dismutase, catalase, peroxidase, and glutathione reductase. Pre-treatment with fluridone significantly suppressed the NaCl-induced increases, but only slightly delayed the increases in tissue subjected to exogenous ABA treatment. This implies that ABA is involved in the signal transduction pathway associated with the NaCl-induced up-regulation of these antioxidant enzymes. Pre-treatment with fluridone had no effect on the paraquat-induced increases, suggesting that these enzymes can also be up-regulated by a pathway other than the one mediated by ABA. Both the NaCl and paraquat treatments produced significant increases in the superoxide levels within the callus, but the increase resulting from the paraquat treatment was significantly higher than the increase resulting from the NaCl treatment. These data suggest that NaCl stress results in the production of reactive oxygen intermediates (ROI) which signals the induction of an ABA-dependent signaling pathway. The production of very high levels of ROI, such as those that occur with paraquat treatment or perhaps during periods of prolonged or extreme stress, may induce an ABA-independent signaling pathway.