The optimization of operating parameters for the isolation of peroxidase from horseradish (Armoracia rusticana) roots with ultrafiltration (UF) technology was systemically studied. The effects of UF operating conditions on the transmission of proteins were quantified using the parameter scanning UF. These conditions included solution pH, ionic strength, stirring speed and permeate flux. Under optimized conditions, the purity of horseradish peroxidase (HRP) obtained was greater than 84 % after a two-stage UF process and the recovery of HRP from the feedstock was close to 90 %. The resulting peroxidase product was then analysed by isoelectric focusing, SDS–PAGE and circular dichroism, to confirm its isoelectric point, molecular weight and molecular secondary structure. The effects of calcium ion on HRP specific activities were also experimentally determined.

In this study, we explored how ammonium and metal ion stresses affected the production of recombinant hyperthermostable manganese superoxide dismutase (Mn-SOD). To improve Mn-SOD production, fed-batch culture in shake flasks and bioreactor fermentation were undertaken to examine the effects of \( {\text{NH}}_{ 4}^{{^{ + } }} \) and Mn2+ feeding. Under the optimized feeding time and concentrations of \( {\text{NH}}_{ 4}^{{^{ + } }} \) and Mn2+, the maximal SOD activity obtained from bioreactor fermentation reached some 480 U/ml, over 4 times higher than that in batch cultivation (113 U/ml), indicating a major enhancement of the concentration of Mn-SOD in the scale-up of hyperthermostable Mn-SOD production. In contrast, when the fed-batch culture with appropriate \( {\text{NH}}_{ 4}^{{^{ + } }} \) and Mn2+ feeding was carried out in the same 5-L stirred tank bioreactor, a maximal SOD concentration of some 450 U/ml was obtained, again indicating substantial increase in SOD activity as a result of \( {\text{NH}}_{ 4}^{{^{ + } }} \) and Mn2+ feeding. The isoelectric point (pI) of the sample was found to be 6.2. It was highly stable at 90 °C and circular dichroism measurements indicated a high α-helical content of 70 % as well, consistent with known SOD properties. This study indicates that \( {\text{NH}}_{ 4}^{{^{ + } }} \) and Mn2+ play important roles in Mn-SOD expression. Stress fermentation strategies established in this study are useful for large-scale efficient production of hyperthermostable Mn-SOD and may also be valuable for the scale-up of other extremozymes.

Our previous work has demonstrated the isolation of photosystem I (PSI) from spinach using ultrafiltration with a final purity of 84 %. In order to get a higher purity of PSI and more importantly to develop a practical bioseparation process, key physiochemical properties of PSI and their dependence on operational parameters must be assessed. In this study, the effect of solution pH, one of the most important operating parameters for membrane process, on the property of PSI was examined. Following the isolation of crude PSI from spinach using n-dodecyl-beta-d-maltoside as detergent, the isoelectric point, aggregation size, zeta potential, low-temperature fluorescence, atomic force microscopy imaging, secondary structure, and thermal stability were determined. Solution pH was found to have a significant effect on the activity, aggregation size and thermal stability of PSI. The results also suggested that the activity of PSI was related to its aggregation size.

The isolation of photosystem-I (PS-I) from spinach has been conducted using ultrafiltration with 300 kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on PS-I activity were optimized using parameter scanning ultrafiltration. These conditions included solution pH, ionic strength, stirring speed, and permeate flux. The effects of detergent (Triton X-100 and n-dodecyl-beta-D-maltoside) concentration on time dependent activity of PS-I were also studied using an O2 electrode. Under optimized conditions, the PS-I purity obtained in the retentate was about 84% and the activity recovery was greater than 94% after ultrafiltration. To our knowledge, this is the first report of the isolation of a membrane protein using ultrafiltration alone.

The optimization of operating conditions for cellulose hydrolysis was systemically undertaken using an ultra-scaled down membrane bioreactor based on the parameter scanning ultrafiltration apparatus. The bioconversion of cellulose saccharification was carried out with freely suspended cellulase from Aspergillus niger as the biocatalyst. The polyethersulfone ultrafiltration membranes with a molecular weight cutoff of 10 kDa were used to construct the enzymatic membrane bioreactor, with the membrane showing a complete retaining of cellulase and cellobiase. The influence of solution pH, temperature, salt (NaCl) concentration, presence of cellobiase, cellulose-to-enzyme ratio and stirring speed on reducing sugar production was examined. The results showed that the addition of an appropriate amount of NaCl or cellobiase had a positive effect on reducing sugar formation. Under the identified optimal conditions, cellulose hydrolysis in the enzymatic membrane bioreactor was tested for a long period of time up to 75 h, and both enzymes and operation conditions demonstrated good stability. Also, the activation energy (Ea) of the enzymatic hydrolysis, with a value of 34.11 ± 1.03 kJ mol−1, was estimated in this study. The operational and physicochemical conditions identified can help guide the design and operation of enzymatic membrane bioreactors at the industrial scale for cellulose hydrolysis.

The objective of this work is to develop a membrane-based cost-effective process for the separation and purification of superoxide dismutase (SOD) from garlic. It was found that a two-stage ultrafiltration process using a 100 kDa MWCO regenerated cellulose membrane and a 50 kDa MWCO polyethersulfone membrane could successfully be employed to isolate SOD by removing the bigger macromolecules and smaller molecules from the crude feedstock. The effects of solution pH and ionic strength on the transmission of SOD and the presence of impurity proteins with molecular weights close to SOD were quantified using the pulsed sample injection ultrafiltration technique. The effect of other parameters including system hydrodynamics and mass transfer, in terms of stirring speed, and permeate flux on the separation of SOD was also studied using parameter scanning ultrafiltration. Under optimized conditions, the purity of SOD obtained was greater than 98.0% after two-stage ultrafiltration process and the recovery of SOD from the feedstock was close to 90%. The resulting SOD product was analyzed by isoelectric focusing (IEF), SDS-PAGE and circular dichroism (CD), to confirm its isoelectric point, molecular weight and molecular secondary structure of the garlic SOD. The purity and activity as well as effects of metal ions (copper and zinc ions) on SOD specific activities were experimentally determined.

Although the importance of eggs as a source of specific antibodies has been well recognised, the generation of egg yolk immunoglobulins (IgY) is rarely chosen due to the peculiar composition of egg yolk and the lack of specific affinity ligands. In this work, we report a novel membrane based two-stage ultrafiltration process to isolate IgY from egg yolk. The effects of solution pH, ionic strength, stirring speed and permeate flux on the transmission of proteins were quantified using the pulsed sample injection technique and parameter scanning ultrafiltration. Under optimised conditions, the purity of immunoglobulin obtained was greater than 93% after the two-stage ultrafiltration process and the recovery of immunoglobulin from the feedstock was close to 87%. The resulting immunoglobulin product was then analysed by Isoelectric Focusing (IEF), SDS–PAGE and Circular Dichroism (CD), to confirm its isoelectric point, molecular weight and molecular secondary structure.