Heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli DH5alpha at 37 degrees C for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at 20 degrees C in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature (Tt) of levansucrase-ELP[V-40] was 17 degrees C with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.     

On-line separation of solute mixtures using reciprocating size exclusion chromatography

Solutes of different size in a mixture solution were separated on-line, using a semi-continuous reciprocating size exclusion chromatography. The band of fast-moving large molecules was isolated during the first half cycle, while the band of slow-moving small molecules was isolated during the second half cycle. After 7 cycles of frontal mode operation, 89% of the Blue Dextran in the feed was isolated as a pure solution. Vitamin B₁₂ of constant concentration was also isolated as a pure solution.     

Concentration of high molecular weight solute by swelling of hydrogel

Summary The size exclusion effect produced by the swelling of hydrolyzed polyacrylamide gel was exploited to concentrate a high molecular weight solute in batch. Changing the solvent composition induced gel swelling or shrinking. Several solutes with different molecular weights were used to test the degree of hydrogel exclusion.     

Recovery of β-Galactosidase from E. Coli Double Lysogen Using Aqueous Two Phase System

An aqueous two phase system (ATPS) was employed to recover -galactosidase from the lytic broth of the phage double lysogen system, where production of an intracellular product and cell disruption are carried out sequentially in a container. Partition of -galactosidase was more favorable to PEG phase comparing to total proteins, which was enhanced with Na₂SO₄. Recovery of -galactosidase was more than 90% in most cases, which was much more the recovery of total proteins.     

Separation of lactic acid from acetic acid using a four-zone SMB

A simulated moving bed (SMB) process has been developed to separate l-(+)-lactic acid from acetic acid, a major impurity in the fermentation broth of Lactobacillus rhamnosus. Poly(4-vinylpyridine) resin (PVP) was selected as the adsorbent. Adsorption isotherms and mass transfer parameters of the organic acids were estimated from single-column frontal tests. Experimental results show that the Langmuir isotherms obtained from the frontal tests can be used in the design of an SMB process to achieve 99.9% purity and over 93% yield of lactic acid. The column profiles and effluent histories, however, deviate from rate model predictions based on the Langmuir isotherms. They agree more closely with the predictions based on a modified Langmuir isotherm for lactic acid. The standing wave design method for systems with modified Langmuir isotherms is developed in this study. Rate model simulations show that the process based on the modified design method can achieve high purity (>99.9%) and high yield (>99.9%). For this nonlinear system, accurate isotherm model and model parameters are needed in the design, and the zone flow rates must be closely monitored and controlled in order to ensure high purity and high yield in the SMB process.     

Modeling of typical microbial cell growth in batch culture

A mathematical model was developed, based on the time dependent changes of the specific growth rate, for prediction of the typical microbial cell growth in batch cultures. This model could predict both the lag growth phase and the stationary growth phase of batch cultures, and it was tested with the batch growth ofTrichoderma reesei andLactobacillus delbrucckii.     

Plant terpenes and lignin as natural cosubstrates in biodegradation of polyclorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs)

The objective of this minireview is to examine how cometabolic biodegradation of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) might be affected by plant terpenes and lignins as natural substrates abundant in nature. The topics covered, hence, are environmental significance of PCBs and PAHs, nature and distribution of plant terpenes and lignin, structural and metaoblic similarities of the natural compounds to PCBs and PAHs, and possible roles of the natural substrates in inducing the biodegradative pathways of PCBs and PAHs.     

Enantioselective production of levofloxacin by immobilized porcine liver esterase

Porcine liver esterase, which cleaves ofloxacin butyl ester enantioselectively to levofloxacin, was successfully immobilized in calcium alginate and polyacrylamide gel. Immobilized esterase in 5% (w/v) calcium alginate exhibited 58% immobilization efficiency and could be reused five times without severe loss of enzyme activity. On the other hand, entrapped esterase in polyacrylamide gel, composed of 20% of total monomer and 8.3% of cross-linking agent, could be reused 10 times, and 51% of enzyme activity remained after the 10th batch without decrease of enantioselectivity. Compared with entrapped methods, significant reduction of enzyme activity was found in the case of physical adsorption on to QAE-Sephadex.