Application of mixed modal resin for purification of a fibrinolytic enzyme.

Recent advances in purification technologies for therapeutic molecules have stirred the research consortium. Mixed mode chromatography, having multiple interactions with the solute molecule, has drawn significant attention due to its overall advantage over traditional ion-exchange and reverse-phase chromatography. Capto adhere, a mixed mode chromatography resin with strong anion-exchange and reverse-phase interaction with solutes, was explored for purification of fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Static and dynamic resin binding study revealed that 30°C temperature, pH 8, and 0.5 mL/min flow rate were optimum for maximum binding of fibrinolytic enzyme. Maximum static dynamic binding and breakthrough capacities for Capto adhere were 249 and 196 U/mL of resin, respectively. Final purification with Sephadex G 100 gel chromatography resulted in 38-fold purity of fibrinolytic enzyme with 39% enzyme recovery. Purified enzyme was further characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis to homogeneity, and molecular mass was found to be around 55-70 kD. Like most of the serine alkaline proteases, purified fibrinolytic enzyme was stable in a temperature range of 25-40°C and pH range of 7-9. Offshoots of our research findings have revealed a broad application area of mixed mode chromatography.

Ultrasound assisted production of a fibrinolytic enzyme in a bioreactor.

The present work illustrates ultrasound assisted production of a fibrinolytic enzyme at 1L bioreactor scale from Bacillus sphaericus MTCC 3672. To alleviate the shortcomings of one factor at a time method of optimization, central composite rotatable design of response surface methodology was employed for optimization of ultrasound assisted production. Different process parameters such as irradiation time, duty cycle and power of ultrasound were varied in 3 different levels in 11 experimental runs. For evaluating mass transfer enhancement effect of ultrasonication on production, control non sonicated fermentation was optimized by varying different agitation speed (300-500rpm) and aeration rate (8.33-33.33cc/s). Optimized ultrasonication protocol resulted in 1.48-fold increase in fibrinolytic enzyme yield as compared to non sonicated fermentation, which comprised of ultrasound irradiation at 25kHz for 10min with 40% duty cycle and 160W power on 12h of growth phase in 1L bioreactor operated at 400rpm agitation speed and 16.66cc/s aeration rate. Declined glucose concentration from 0.1% w/v (non sonicated control run) to 0.05% w/v and breakage of cells cluster emphasized on increased substrate utilization potential and enhanced convection of ultrasound assisted fermentation in a bioreactor. Deliverables of current studies will provide significant insights for enhancement of productivity of various enzymes at a bioreactor level.

Ultrasound stimulated production of a fibrinolytic enzyme.

The present study is aimed at enhanced production of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672 under ultrasonic stimulation. Various process parameters viz; irradiation at different growth phases, ultrasonication power, irradiation duration, duty cycle and multiple irradiation were studied for enhancement of fibrinolytic enzyme productivity. The optimum conditions were found as follows, irradiation of ultrasonic waves to fermentation broth at 12 h of growth phase with 25 kHz frequency, 160 W ultrasound power, 20% duty cycle for 5 min. The productivity of fibrinolytic enzyme was increased 1.82-fold from 110 to 201 U/mL compared with the non sonicated control fermentation. Drop in glucose concentration from 0.41% to 0.12% w/v in ultrasonicated batch implies that, ultrasonication increases the cell permeability, improves substrate intake and progresses metabolism of microbial cell. Microscopic images before and after ultrasonic stimulation clearly signifies the impact of duty cycle on decreasing biomass concentration. However, environmental scanning electron micrograph does not show any cell lysis at optimum ultrasonic irradiation. Offshoots of our results will contribute to fulfill the demand of enhancement of microbial therapeutic enzyme productivity, through ultrasonication stimulation.

Ultrasound assisted three phase partitioning of a fibrinolytic enzyme.

The present investigation is aimed at ultrasound assisted three phase partitioning (UATPP) of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Three phase partitioning integrates the concentration and partial purification step of downstream processing of a biomolecule. Three phase system is formed with simultaneous addition of ammonium sulfate to crude broth and followed by t-butanol. UATPP of a fibrinolytic enzyme was studied by varying different process parameters such as ammonium sulfate saturation concentration, pH, broth to t-butanol ratio, temperature, ultrasound frequency, ultrasonication power, and duty cycle. The optimized parameters yielding maximum purity of 16.15-fold of fibrinolytic enzyme with 65% recovery comprised of 80% ammonium sulfate saturation, pH 9, temperature 30 °C, broth to t-butanol ratio 0.5 (v/v), at 25 kHz frequency and 150 W ultrasonication power with 40% duty cycle for 5 min irradiation time. SDS PAGE analysis of partitioned enzyme shows partial purification with a molecular weight in the range of 55-70 kDa. Enhanced mass transfer of UATPP resulted in higher fold purity of fibrinolytic enzyme with reduced time of operation from 1 h to 5 min as compared to conventional TPP. Outcome of our findings highlighted the use of UATPP as an efficient biosepartion technique.