When participating in the Asia Pacific Biochemical Engineering Conference (APBioChEC) in Singapore in 1994, I was fortunate to attend a workshop seminar given by Prof. Zhiguo Su from Dalian University of Technology, Dalian, PRC. The title of the seminar was “Bioseparation in the Peoples Republic of China” in which he displayed a profound knowledge of the current situation for biotechnology in his homeland. After the seminar I approached Prof. Su introducing myself. We immediately realized that we shared a strong interest in protein purification and the same year he invited me to give a lecture to his students and staff at his department. Soon after, in 1997, Prof. Suwas appointedDeputyDirector of the State Key Laboratory of Biochemical Engineering in the Institute of Process Engineering at the Chinese Academy of Sciences in Beijing and promoted to its Director in 2001. In 2013 till date, Prof. Su has been Chief Scientist at the National Engineering Research Center for Biotechnology in Beijing. During all these years we have kept a close contact for joint research in the area of bioseparation. Bioseparation is a key support area of research for the development of modern life science and technology. In scientific laboratories, biomolecules are separated to ensure a pure substance for further study. In industry, various biological products, such as blood proteins, vaccines and recombinant pharmaceuticals, have to be purified to guarantee safety, activity and long term stability. Despite its importance, not many researchers would like to engage in bioseparation research because it involves multidisciplinary knowledge and know-how experience. Furthermore, it is not an area generating publications in journals of high impact factors. Professor Su and I share a strong interest in bioseparation because we both realize its power in solving practical problems both in life science laboratories and in industry. For more than 20 years, we have collaborated on the path of innovation. Here, I would like to introduce some of our research results and corresponding techniques.

A strain of Bacillus subtilis FS2 isolated from a traditional fish source in Vietnam has been shown to produce an enzyme possessing both a gelatinase and a lipase A (LipA) activity when tested on agar plates containing 0.1% tributyrin and 0.3% gelatine. A gene encoding LipA was isolated and expressed in E. coli BL21 (DE3). The enzyme has got an apparent Mr of 24 kDa, a pI of 9.2 and was purified to electrophoretical homogeneity by affinity chromatography. Both the lipase and the gelatinase showed optimum activity at pH 10 and at 30 0 C, with highest activity in the range 25-37°C. The metal ions Fe++, Co++, and Mn++ at 10 mM had strong inhibitory effect on both activities whereas Ca++ had no effect and Zn++ had a slight enhancing effect. The specific enzymatic activities obtained were 19814 U/mg for the lipase and 1245 U/mg for the gelatinase.

The SPG (Shirasu porous-glass) membrane emulsification technique has been subject to much attention for the preparation of uniform emulsions. However, so far primarily used for the production of droplets with sizes below approximately 60μm. A production bottleneck occurred if the desired size was further increased, especially when highly viscous dispersed phases were involved. To this end, an improved membrane emulsification technique was proposed and has been applied to the preparation of large agarose microspheres, with a size of around 90μm and with a narrow size distribution. The effects of important emulsification parameters, including the pore size of the SPG membrane, the operating pressure, the stirring rate of the continuous phase, the composition of the continuous oil phase, and the concentration of agarose in the dispersed water phase, have been extensively studied. Under optimum conditions, uniform-size agarose microspheres with an average diameter of 93μm and a size distribution index of 0.65 were successfully prepared. The average particle size of the home-made agarose microspheres was almost identical to that of the commercial product Sepharose 4 Fast Flow (4FF), which is produced by mechanical stirring and an additional sieving process. However, the size distribution of the former was much narrower than that of the latter. Therefore, the improved membrane emulsification technique presented here is promising for the application of high viscosity systems such as agarose solutions, and the production scale can be further enhanced by increasing the number of membrane units attached to the experimental apparatus.

High‐speed counter‐current chromatography using a new liquid–liquid/solid three‐phase system was used for the separation of the polyphenols corilagin and geraniin from a crude extract of G eranium wilfordii M axim in one step. The optimized three‐phase system was composed of n ‐hexane/ethyl acetate/methanol/acetic acid/water and to which was added 10‐μm average diameter microspheres of cross‐linked 12% agarose at the ratio of 0.2:10:2:1:5 and 0.1 g/mL, respectively. The purities of geraniin and corilagin were 82 and 90%, which were determined by HPLC at 280 nm. A 14.5 and 7 mg of geraniin and corilagin were purified from 160 mg crude extract with the yields of 70 and 78%, respectively.

Protein–ligand interactions on liquid–solid interfaces governed the design of functional biomaterials. However, accurate residue details of ligand induced protein binding and unfolding on an interface were still unknown by the current ensemble of protein structure characterizations. Here, a hydrogen/deuterium (H/D) approach coupled with analysis of NMR TOCSY spectra and the solvent accessible surface area (SASA) was designed to enable residue level understanding of lysozyme adsorbed at a phenyl-ligand modified surface. Results showed that the binding sites and unfolding of lysozyme molecules on phenyl-agarose microspheres demonstrated significant ligand-density dependence and protein-coverage dependence. Either increasing ligand density or decreasing adsorption coverage would lead to more binding sites and unfolding of the protein molecules. With the multipoint adsorption strengthening, the protein molecule changed from lying end-on to side-on. Finally, Molecular Dock simulation was utilized to evaluate the NMR determined binding sites based on energy ranking of the binding. It confirmed that this NMR approach represents a reliable route to in silico abundant residue-level structural information during protein interaction with biomaterials.

Dextranases catalyze the hydrolysis of the α-l,6-glucosidic bond of the polysaccharide dextran. Dextranases have been isolated from bacteria, yeast and fungi. Purified dextranase enzyme from Penicillium sp. was PEGylated (polyethylene glycol modification) with mPEG (5000Da) and showed an increase in the dextranase protein molecular weight as estimated by Superose 12 (23ml) column and this increment in the molecular weight is directly proportional to mPEG (5000Da) concentration until a complete dextranase enzyme PEGylation (disappearance of dextranase peak). The residual activity of partially PEGylated dextranase (mPEG 5000 of 5.8mg/ml) was 33.8% and for the completely PEGylated dextranase (mPEG 5000 of 29mg/ml) it was 25.75%. Dextranase PEGylated with mPEG (30,000Da) showed a little PEGylation at mPEG concentration of 5.8mg/ml but at a concentration of 29mg/ml several PEGylated peaks were produced with a difference in dextranase activity toward dextran T500, retardation in the activity with the increasing in the molecular weight was clearly appeared with Sephadex G75 but for Sephadex G200 a little retardation than Sephadex G75 has been appeared.

This chapter contains sections titled: Introduction The Protein Extract An Overview of Fractionation Techniques Fractionation Strategies Monitoring the Fractionation The Final Product Laboratory Equipment References

The hydrolysable tannins corilagin and geraniin, the major active components of the traditional Chinese medicine Geranium wilfordii Maxim, have been separated and purified from crude extracts in one step by adsorption chromatography on cross-linked 12% agarose gel (Superose 12 10/300 GL). The separation was achieved by gradient elution using mobile phase A composed of 5% ethanol and 5% acetic acid and mobile phase B composed of 30% ethanol and 30% acetic acid. The gradients were composed as follows: 0-240 mL, 0-25% B; 240-480 mL, 25-40% B; after 480 mL, 100% B. The purities of the collected corilagin and geraniin were 92.4 and 87.2%, and the corresponding yields were 88.0 and 76.8%, respectively.

This chapter contains sections titled: Introduction: Basic Concepts and Versions of Chromatography The Stationary Phase Chromatographic Theory Chromatographic Procedures Chromatographic Techniques On The History of Protein Chromatography References

This chapter contains sections titled: Introduction Affinity Interactions Preparation and Evaluation of Affinity Adsorbents Immobilization Techniques Chromatographic Techniques Applications References

A porous polyamide resin is shown to possess hydrogen bond acceptor properties suitable for the separation of polyphenolic solutes such as phenolic acids, flavonols and flavonoids. The separation is achieved in the presence of solvent mixtures of acetic acid and ethanol. The extent of hydrogen bond adsorption is reviewed based on data obtained from the elution behaviour of a variety of simple polyphenolic solutes. Polyamide adsorption chromatography was applied for the purification of resveratrol and polydatin from Polygonum cuspidatum Sieb. & Zucc.

[Objective]The research aimed to study the separation and purification of mussel adhesive protein by extensive cross-linking agarose based multimodal gel and discuss the separation mechanism.[Method]Adsorption and desorption behaviors between the gel and the protein were studied by using static and dynamic adsorption method under different salt concentrations and different potential of hydrogen.[Result]The salt concentration increased from 0 to 0.5 mol/L.The changes of equilibrium adsorption capacity between media and protein was within 6% .The alkalinity acidity had greater effects on the adsorption.When the acidity was across the isoelectric point of the protein,the change percentage of the maximum adsorption per pH unit was up to 23% .[Conclusion]The multimodal adsorption between the multimodal ligand of the gel and mussel adhesive protein involves ionic interaction,hydrophobic interaction and hydrogen bond adsorption.When the eluent with the acidity of higher than the isoelectric point was used to elute the mussel adhesive protein,the mussel adhesive protein with the puri-ty of 90% was obtained.

Following its market introduction in 1982, the cross-linked 12% agarose gel media Superose 12 has become widely known as a tool for size exclusion chromatography of proteins and other biological macromolecules. In this review it is shown that, when appropriate mobile phases are used, Superose possesses adsorption properties similar to that of traditional media for hydrophilic interaction liquid chromatography (HILIC). This is illustrated by the separation and purification of low molecular weight compounds such as polyphenols including active components of traditional Chinese medicinal herbs and green tea. Structural features of the cross-linked agarose that likely cause the observed adsorption effects are discussed as well. These are identified as being primarily ether bonds acting as strong hydrogen bond acceptors as well as hydrophobic residues originating from the cross-linking reagents.

The highly cross-linked 12% agarose gel Superose 12 10/300 GL causes retardation of glycine peptides when mobile phases containing varying concentrations of acetonitrile in water are used. An investigation has been made into the retention mechanism behind this retardation using the glycine dipeptide (GG) and tripeptide (GGG) as models. The dependence of retention times of analytical-size peaks under different experimental conditions was interpreted such that the adsorption most probably was caused by the formation of hydrogen bonds but that electrostatic interactions cannot be ruled out. Thereafter, a nonlinear adsorption study was undertaken at different acetonitrile content in the eluent, using the elution by characteristic points (ECPs) method on strongly overloaded GG and GGG peaks. With a new evaluation tool, the adsorption energy distribution (AED) could be calculated prior to the model selection. These calculations revealed that when the acetonitrile content in the eluent was varied from 0% to 20% the interactions turned from (i) being homogenous (GG) or mildly heterogeneous (GGG), (ii) via a more or less stronger degree of heterogeneity around one site to (iii) finally a typical bimodal energy interaction comprising of two sites (GG at 20% and GGG at 10% and 20%). The Langmuir, Tóth and bi-Langmuir models described these interesting adsorption trends excellently. Thus, the retardation observed for these glycine peptides is interpreted as being of mixed-mode character composed of electrostatic bonds and hydrogen bonds.

Surfactant-catalyzed room-temperature radical polymerization of methyl methacrylate (MMA) was conducted in the presence of fumed silica nanoparticles and water. Three types of surfactants, cationic (CTAB), nonionic (Triton X-100) or anionic (SDS), were used to catalyze the decomposition of the initiator, 2,2′-azobisisobutyronitrile (AIBN). The surfactant-catalyzed decomposition rate constant for AIBN at room temperature was found to be independent of the surfactant type. However, the rates of polymerization of the MMA emulsion gels at room temperature were found to depend on the types of surfactant with: cationic > nonionic > anionic. An inhibition period was observed for the polymerizations with nonionic and anionic surfactants. The radical-inhibition was likely due to the reactions between the radicals and the silanol groups on fumed silica. This inhibition can be reduced by using cationic surfactants to block these surface silanols.