Xuejun Cao

Concentration of 6-aminopenicillanic acid from penicillin bioconversion solution and its mother liquor by nanofiltration membrane

In this study, nanofiltration was applied to the concentration of the 6-aminopenicillinic acid (6-APA) from bioconverted penicillin solution and also to its mother liquor. The 6-APA in the solution was concentrated from 0.211 mol/L to 0.746 mol/L by nanofiltration. The final maximum concentration was 3.6 times higher than the initial concentration and the recovery yield was 97% to 99% of the original 6-APA. The concentrated solution was crystallized with the yields of 88.9–90.2% and the purity of the crystallized product was about 98%. The concentration of 6-APA in the mother liquor after crystallization was 0.014 mol/L and thus was concentrated 20–30 fold by nanofiltration and crystallization. The recovery of 6-APA was over 98%. The salts contained in the mother liquor, such as NH4Cl and KCl, could be removed by allowing them to permeate through the membrane.

Application of docking methods for metal chelate affinity precipitation of endo-glucanase using pH-response polymer.

An endo-glucanase could be efficiently purified using metal chelate affinity precipitation by a pH-response polymer PMMDN with iminodiacetic acid (IDA) and Cu(2+) as affinity ligand. In this study, docking method was used to identify the appropriate chelator and the metal ion as ligand by Grid score. The simulation results were compared with the label-free detection data analyzed by ForteBios Octet. The ligand IDA-Cu(2+) was the final choice. A pH-response polymer PMMDN was polymerized and subsequently coupled with IDA-Cu(2+)as the ligand. The pI and recovery of PMMDN and PMMDN-IDA-Cu(2+) were 4.50, 99.8% and 4.39, 97.6%, respectively. Optimal adsorption conditions were found to be ligand density of 3.0 mmol/g, pH 5.5 and 1.0 mol/L NaCl. The adsorption isotherm showed the maximum adsorption as 57.62 mg/g polymer and the dissociation constant as 1.08 mg/mL. For the elution of the PMMDN-IDA-Cu(2+) with the protein, 0.5 mol/L imidazole containing 1.0 mol/L guanidine hydrochloride was used as the eluent. Under these conditions, electrophoretic purity of endo-glucanase was obtained by only one step, and the elution recoveries were 96.45% (protein) and 93.24% (activity).

Lipase purification by affinity precipitation with a thermo-responsive polymer immobilized Cibacron Blue F3GA ligand

A thermo-responsive polymer (PNNB) was synthesized with lower critical solution temperature 27.5°C and over 95% recovery. The adsorption of porcine pancreatic lipase on Cibacron Blue F3GA-conjugated PNNB (PNNB-CB) closely followed the bi-Langmuir adsorption isotherm. The maximum adsorption capacity was found at pH 5.0, with a ligand density of 18.4 μmol/g polymers. The optimized eluent was a 0.01 M phosphate buffer solution at pH 8.0 containing 20% ethylene glycol. Six adsorptiondesorption recycles indicated excellent reusability of the affinity adsorbent. PNNB-CB was applied to separate porcine pancreatic lipase from its crude material giving a lipase activity recovery of 81.6% with a 16-fold purification factor. Lipase could be purified to single-band purity, according to gel electrophoresis. The purification strategy is therefore feasible and efficient for purifying proteins of interest.

Development of pH-responsive polymer and citrate aqueous two-phase system for extractive bioconversion of cefprozil

A pH-responsive aqueous two-phase system (pH-ATPS) has been developed by sodium citrate and a recyclable pH-responsive polymer PADB6.8 that can response to the change of pH values. Phase separation mechanism is studied through Low field-NMR. All variables affecting the phase separation are evaluated. Phase characteristics (viscosity, density, interfacial tension) and phase separation kinetic are studied for understanding of separation process and operational parameters in applications. This pH-ATPS has the characters of low interfacial tension, high recovery leading efficient mass transfer and low cost. The proposed system can be used as a mild medium for extractive bioconversion with low cost. We applied this pH-ATPS in extractive bioconversion of cefprozil. Cefprozil is partitioned towards the polymer-rich phase while the substrates tended to be partitioned in the salt-rich phase. Extractive bioconversion of cefprozil in this pH-ATPS can improve yield of the enzymatic process and reduce the product hydrolysis in optimal conditions. The maximal conversion yield of cefprozil in the studied system is 91.0%.

Biodegradation of cellulose by β-glucosidase and cellulase immobilized on a pH-responsive copolymer

Biodegradation of cellulose involves synergistic action of the endoglucanases, exoglucanases and β-glucosidases in cellulase. However, the yield of glucose is limited by the lack of β-glucosidase to hydrolyze cellobiose into glucose. In this study, β-glucosidase as a supplemental enzyme along with cellulase are co-immobilized on a pHresponsive copolymer, poly (MAA-co-DMAEMA-co-BMA) (abbreviated PMDB, where MAA is α-methacrylic acid, DMAEMA is 2-dimethylaminoethyl methacrylate and BMA is butyl methacrylate). The thermal and storage stabilities of PMDB with immobilized enzymes are improved greatly, compared with those of free cellulase. Biodegradation of cellulose is carried out in a pH-responsive recyclable aqueous two-phase system composed of poly (AA-co- DMAEMA-co-BMA) (abbreviated PADB 3.8, where AA is acrylic acid) and PMDB. Insoluble substrate and PMDB with immobilized cellulase and β-glucosidase (Celluclast 1.5L FG and Novozyme 188, respectively) were biased to the bottom phase, while the product was partitioned to the top phase in the presence of 40 mM (NH4)2SO4. When the degradation reaction of cellulose is carried out with PMDB containing immobilized cellulase and β-glucosidase, the concentration of glucose reaches 4.331 mg/mL after 108 h. The yield of glucose is 50.25% after PMDB containing the immobilized enzymes is recycled five times.

Synthesis of cefprozil using penicillin G acylase in recyclable aqueous two-phase systems

Cefprozil is an important semi-synthetic cephalosporin antibiotic. In this study, immobilized penicillin G acylase (PGA) is used to catalyze the acylation of 7- amino-3-(1-propenyl)-4-cephalosporanic acid (7-APRA) and 4-hydroxyphenylglycine methyl ester (HPGME) and a recyclable thermo-pH responsive PNB/PADB aqueous twophase system (ATPS) is used to synthesize cefprozil. In this system, the partition coefficient of cefprozil was 2.24 with 60 mmol/L (NH4)2SO4. In addition, the optimal enzymatic reaction conditions were found to be pH 6.5, 20°C, 78 u/mL immobilized PGA, 30 mmol/L 7-APRA and 90 mmol/L HPGME. In the PNB/PADB ATPS, the maximal yield of cefprozil was 75.81% with 60 mmol/L (NH4)2SO4 and in the single aqueous system the yield was 56.02%. The yields are thought to improve because there is a reduction in product inhibition.

Recyclable aqueous two-phase system based on two pH-responsive copolymers and its application to porcine circovirus type 2 Cap protein purification

Aqueous two-phase system (ATPS) has great potential in industrial applications of bio-separations and bio-reactions. However, its large-scale application is limited by recovery difficulty of phase systems. In this paper, a recyclable ATPS was prepared by two pH-responsive copolymers (PADB4.99 and PMDM7.08) and applied for purification of porcine circovirus type 2 Cap protein fermentation broth (PCV2 Cap protein). Phase separation mechanism was studied by using low-field nuclear magnetic resonance (LF-NMR). The results showed that relatively desirable ATPS were formed successfully when two copolymer concentrations were 4%-6% (w/w) with pH 7.5-8.6. The main parameters, such as copolymer concentration, temperature, pH, type and concentration of salts, were investigated. The results demonstrated that the best ATPS consisted of 4% (w/w) PADB4.99 and 6% (w/w) PMDM7.08, the optimal partition coefficient (K) and extraction recovery (ER) of PCV2 cap protein were 0.25 and 94.2% in the presence of 50 mM Li2SO4, 3.53 and 93.4% in the presence of 40 mM KCl, respectively, at 30 °C, pH 8.1. The maximum purity of PCV2 Cap protein was 88.4%. In addition, the two phase-forming copolymers could be recycled by adjusting solution pH to their isoelectric points (pI), with recoveries of over 97.5%.

Preparation and Characterization of a pH-responsive Polymer that Interacts with Microbial Transglutaminase during Affinity Precipitation

Microbial transglutaminase (MTG) has been widely used in the food and pharmaceuticals industries. In this study, MTG was purified using affinity precipitation with an affinity polymer (PMMDN-T), which was synthesized using a pH-responsive polymer (PMMDN) coupled with L-thyroxin as an affinity ligand. Interactions between MTG and PMMDN-T were investigated using turbidimetric titration, zeta potential measurements, and low-field nuclear magnetic resonance (LF-NMR). We found different behaviors, architectures, and phase states of pH-dependent interactions between MTG and PMMDN-T interactions. Binding energetics between MTG and PMMDN-T were determined by isothermal titration calorimetry (ITC). The isoelectric point (pI) of the affinity polymer was 4.65 and was recovered with 96.7% efficiency after recycling the polymer three times. The optimal adsorption condition was 0.02 mol/L phosphate buffer (pH 6.0) with 1.0 mol/L NaCl at 30.0°C and a ligand density of 50.0 μmol/g. The maximum elution recoveries of total MTG were 98.44% (protein) with 92.19% (activity) using 0.02 mol/L pH 10.0 Gly-NaOH as the eluent.