Investigation Of Enzymatic Activity Of Pretreatment Enzymes In Supercritical CO₂ Fluid And Their Application

The objective of this work is to investigate the enzymatic activity of pretreatment enzymes in supercritical carbon dioxide media, which arefrequently used in biological desizing and/or pretreatment for textiles. Moreover, these employed enzymes are also applied to the pretreatment of cotton fabric in supercritical CO₂ fluid, which could enhance the enzyme activities and the efficiency of the pretreatment, as well as to reduce the utilized amounts of chemicals, decrease the energy consumptions and COD/BOD values of wastewater, shorten the process and save the cost of production. There are great significances with this subject in the protection of environment, the reduction of pollutants emission and energy saving in dyeing and finishing industry.The effects of pressure, temperature and processing time of supercritical CO₂ fluid on pectinase, lipase, cellulase, hemicellulase, laccase and B8360 protease activities were discussed. Furthermore, the primary, secondary and tertiary structures of pectinase whose enzyme activity was significantly changed were investigated by Fourier transform infrared spectroscopy, circular dichroism spectrum, Uv-vis absorption spectroscopy and fluorescence spectrometry. In addition, the application of the one bath process with enzymes and hydrogen peroxide in supercritical CO₂ fluid with grey cotton fabrics was attempted by employing a microemulsion consisted of complex enzymes（mixed by alpha-amylase, pectinase, lipase and hemicellulase）, hydrogen peroxide and supercritical CO₂ fluid. The influences of temperature of supercritical CO₂ fluid, processing time, concentration of complex enzyme and hydrogen peroxide on weight loss rate, capillary effect, whiteness and tensile strength of cotton fabrics were discussed through the analysis of orthogonal design, and optimum processe of pretreatment was selected. Finally, the chemical and aggregation structure changes of the fabric treated by optimum processing were analyzed by Fourier transform infrared spectroscopy and X-ray powder diffraction analysis. Besides, the surface morphology and water absorption capacity of the fabric were studied by scanning electron microscopy and contact angle measurement.The results show that the supercritical CO₂ fluid processing has different effects on activities of different enzymes. The activity of pectinase was improved under appropriate conditions, which could be increased by 37%. With increasing of the pressure, the pectinase activity was increased firstly and then decreased, while it was decreased with the increased temperature and prolonged time of treatment. However, a significant reduction of lipase activity was observed, which was reduced by 50%. Similarly, the lipase activity was also increased with increasing of the pressure, and decreased with increasing temperature and prolongation of treatment time. The cellulase activity could be improved utilizing suitable treatment conditions in supercritical CO₂ fluid, which could be mostly increased by 29%. The activity of cellulase first increased and then decreased with increasing temperature, pressure and treatment time. Moreover, the pressure was the most influential factor for cellulase activity. To a certain extent, the activity of hemicellulase was improved in supercritical CO₂ fluid, which could be increased by 40%. At the same time, hemicellulase activity was enhanced with increasing of pressure and processing time, and decreased with increasing temperature, which was due to the partial inactivation of hemicellulose resulting from low temperature and high pressure. Simultaneously, effect of supercritical CO₂ fluid on activity of laccase was quite remarkable, which could be mostly increased by 47%. Meanwhile, the laccase activity increased first and then decreased with increasing pressure, temperature and treatment time. And the most significant influence on activity of laccase was temperature. In addition, supercritical CO₂ fluid treatment was beneficial to improve the activity of B8360 alkaline protease, and an increase of 23% was observed. The activity of protease showed the trend that first increased and then decreased with increasing of pressure, temperature and treatment time, while there was less effect of the pressure on the activity of protease.The results of structure analyses show that the effects of supercritical CO₂ fluid on different structures of pectinase were diverse. Infrared spectra show that there was no significant change with chemical composition of pectinase applied in supercritical CO₂ fluid pretreatment. Circular dichroism spectra imply that alpha helix structure was the main secondary structure of pectinase, and the activity decreased, along with the obvious increase of random coil content, which could be attributed to the high temperature fluid. Furthermore, beta angle content of the one whose activity enhanced was increased. Uv-vis absorption spectra and fluorescence spectra indicate that the tertiary structure of pectinase changed obviously after pretreatment in supercritical CO₂ fluid, whose absorbance was increased and fluorescence intensity decreased with the increase of pectinase activity.The pretreatment of grey cotton fabrics by employing the one bath process with enzymes and hydrogen peroxide in supercritical CO₂ fluid was perfect feasibility with a better result. The content of H2O₂ was the most significant factor on the weight loss, whiteness and strength of the fabric. At the same time, the capillary effect was greatly influenced by the treatment time. In conclusion, the optimum process based on capillary rise height is recommended as: concentration of complex enzyme at 0.30g/L, volume concentration of hydrogen peroxide at 15ml/L, under the temperature of 55℃ for 4h. While the optimum process based on whiteness is recommended as: enzyme concentration of 0.45g/L, hydrogen peroxide concentration of 15ml/L, under the temperature of 70℃ for 4h.The infrared spectra of cotton fabrics show that the infrared intensities of the main functional groups decreased after the pretreatment in the supercritical fluid, and the peak was shifted to the lower wavenumbers, which indicated that the contents of size and the impurities were reduced. The results of X- ray diffraction suggest that the treatment medium affects the crystal structure of cotton fabric to some extent, the crystallinity was decreased. The results of scanning electron microscopy exhibit that the surfaces of cotton fabrics were severely etched and the size was removed after the pretreatment of cotton fabrics in supercritical CO₂ fluid. The results of contact angle indicate that the water absorption capacities of cotton fabrics were significantly increased with the pretreatment in supercritical CO₂ fluid.