Phase transfer catalyzed bioconversion of penicillin G to 6-APA by immobilized penicillin acylase in recyclable aqueous two-phase systems with light/pH sensitive copolymers / 南方医科大学学报

Immobilized penicillin acylase was used for bioconversion of penicillin PG into 6-APA in aqueous two-phase systems consisting of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB. Partition coefficients of 6-APA was found to be about 5.78 in the presence of 1% NaCl. Enzyme kinetics showed that the reaction reached equilibrium at roughly 7 h. The 6-APA mole yields were 85.3% (pH 7.8, 20 degrees C), with about 20% increment as compared with the reaction of single aqueous phase buffer. The partition coefficient of PG (Na) varied scarcely, while that of the product, 6-APA and phenylacetic acid (PA) significantly varied due to Donnan effect of the phase systems and hydrophobicity of the products. The variation of the partition coefficients of the products also affected the bioconversion yield of the products. In the aqueous two-phase systems, the substrate, PG, the products of 6-APA and PA were biased in the top phase, while immobilized penicillin acylase at completely partitioned at the bottom. The substrate and PG entered the bottom phase, where it was catalyzed into 6-APA and PA and entered the top phase. Inhibition of the substrate and products was removed to result in improvement of the product yield, and the immobilized enzyme showed higher efficiency than the immobilized cells and occupied smaller volume. Compared with the free enzyme, immobilized enzyme had greater stability, longer life-time, and was completely partitioned in the bottom phase and recycle. Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage. The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtered 450 nm light, while pH-sensitive polymer PADB could be recovered at the isoelectric point (pH 4.1). The recovery of the two copolymers was between 95% and 99%.

The influence of controlling redox potential on ethanol production by Saccharomyces cerevisiae / 生物工程学报

Redox electrode was used to control redox potential at four different levels (-50 mV, -100 mV, -150 mV, - 230mV) for the study of ethanol fermentation. The result showed that there was notably influence on the yield of ethanol, the formation of glycerol, the secretion of organic acid, biomass and the death of cell by controlling redox potential at different levels. For example: the biomass of ORP at -50 mV was 1.26, 1.86, 2.59 times higher than ORP at -100 mV, -150 mV, -230 mV respectively, the final glycerol concentration was 1.2, 1.1, 1.7 times higher but final ethanol concentration was 0.87, 0.49, 0.51 times lower compared to the latest three ORP control level. And take biomass, ethanol yield, glycerol concentration, and unconsumed glucose into consider, we get the conclusion that it is very favorable for ethanol fermentation by control ORP at 150 mV. So it give us a apocalypse that we can use redox electrode to control the ethanol fermentation exactly on bioreactor scale.

Phase Transfer Bioconversion of Penicillin G into 6-APA by Immobilized Penicillin Acylase in Recycling Aqueous Two-phase Systems With Light-pH Sensitive Copolymers / 中国生物工程杂志

Immobilized penicillin acylase was used for bioconversion of penicillin G into 6-APA in aqueous two-phase systems consisted of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB.Partition coefficients of 6-APA was found to be:about 5.78,in the presence of 1% NaCl.Enzyme kinetic showed that reaction reached equilibrium at 7h or so.The 6-APA mole yields were 85.3%(pH 7.8,and 20 ℃) and this value was about 20%higher than control in reaction of single aqueous phase buffer.Partition coefficient of penicillin G(Na) washardly changeable,while partition coefficient of product,6-APA and phenylacetate acid was significantly changeable.Reason is due to Donnan effect of phase systems andhydrophobicity of products.The change of partition coefficients of products also affects bioconversion yield of products.In the aqueous two-phase systems,substrate,penicillin G,products 6-APA and phenylacetate acid are biased in top phase,while immobilized penicillin acylase is completely partitioned in bottom.Substrate,penicillin G enters into bottom phase,and it is catalyzed into 6-APA and phenylacetate acid,then the products enter into top phase.Finally,inhibition of substrate and products is removed to result in improvement of products yield.Moreover,immobilized enzymehashigher efficiency than immobilized cells and occupy smaller volume.Comparing with free enzyme,immobilized enzymehashigher stability,longer use life,completely partitioned in bottom phase and recycle.Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage.The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtrated 450 nm light,while pH-sensitive polymer PADB could be recovered by isoelectric point(pH 4.1).The recovery of the two copolymers was 95%~99%.

Over-expression in Escherichia coli and characterization of apolipoprotein AI / 生物工程学报

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.

Quantitation & optimization of guanosine fermentation process: prevention of NH4+ accumulation increases guanosine production by 70% / 生物工程学报

Metabolic engineering has become a powerful tool for optimization of industrial fermentation processes. Metabolic engineering usually undergoes three steps: construction of a recombinant strain with improved properties, genetic and biochemical analysis of the strain, and identification of target for further improvement. Metabolic fluxes analysis is an important part of the biochemical analysis. Based on the law of mass conservation and assuming pseudo-steady-state for the intermediates in the metabolic pathways, we have quantitatively analyzed the time course of the flux distribution in Bacillus subtilis and used the data to reveal the nature of the so-called "40 hour" phenomenon in fermentation of guanosine, a key raw material for the synthesis of additives for human consumption and animal feeds. The phenomenon refers to the observation that guanosine production, which proceeds at high rate from 12 hour on, declines around 40 hour while consumption of glucose keeps increasing, leading to the lower yield of the nucleoside. Equations based upon the metabolic network of Bacillus subtilis consisted of EMP pathway, HMP pathway, TCA cycle, oxidative phosphorylation pathway and others reactions of the intermediates, was constructed. The equations were solved by using the quantitative data obtained in this study. The air flow and volume, concentration of oxygen and carbon dioxide in the exit-gas were monitored online; the concentration of biomass, glucose and guanosine was analyzed manually; and the concentration of acetate, citric acid, pyruvate, and 17 amino acids were HPLC quantified. The solutions of the equation were proved to be valid, as the experimental data on oxygen consumption agrees with that of predicted form the equation. The results indicated that at 40h of the fermentation process the flux of HMP pathway, which provides the precursor of the nucleoside, decreased while that of EMP pathway and the pathways that generate amino acids and organic acids increased. The shift correlated with the accumulation of NH4+ in the broth. The assimilation of NH4+ is an energy consuming process and could shift the metabolism to the energy generating EMP pathway. Accordingly, measures were taken to prevent the accumulation of NH4+. The interference indeed stopped the metabolism shift and boosted the guanosine production at 30 g/L, 70% higher than the level reported in literature.

Quantitative studies of the production phase in rHSA fermentation / 生物工程学报

The model equations of the production phase of rHSA fermentation were derived on the base of both elemental balance and metabolic balance, then the unknown parameters of the model were estimated by multivariable optimization. The possible reasons of discrepancy of production rate between different period of fermentation were discussed. The model could preferably described the relations between different macroscopic reaction rates of the process and keys for the high-efficiency expression of HAS were deduced.

Application of element and metabolism balancing for the cultivation process with Streptomyces aureofaciens / 生物工程学报

On the base of element and metablism balancing, the mathematical model of the cultivation process with Streptomyces aureofaciens was developed, and the unknown parameters in the model were estimated with the method of nonlinear optimization. Firstly the energetic coefficient of CTC biosynthesis was gained, which was 1.8 - 2.8 mol-ATP x C-mol(-1). The macroscopic reaction rates were predicted in the process and compared with the experimental values. The results show that the model can preferably describe the relationships between several macroscopic reaction rates in the process and can supervise the optimization of CTC fermentation process theoretically.

Analysis on metabolic flux shift during guanosine fermentation / 生物工程学报

Taking the typical metabolic control product-guanosine as an example, the method of metabolic flux shift investigation based on process multi-levels parameter correlation analysis was established. The metabolic pathway, multi-parameter correlation, accumulation of amino acid and organic acid during guanosine fermentation process were integratively analyzed. The metabolic flux shift from HMP to EMP was ascertained, which was assumed to be caused by the accumulation of ammonium ion. The subsequent optimization based on controlling flux distribution between EMP and HMP did improve the yield by 35% when the metabolic flux shift was prevented.

High-density Expression of Recombinant Porcine Insulin Precursor by Pichia pastoris / 微生物学通报

The recombinaut porcine insulin precursor(PIP)produced by Pichia pastoris in shake-flask and 501.fermenter was investigated respectively.The results indicated that 60h induction time length and 2.0%～2.5% methanol addition every day was optimum in shake- flask.The process in 50L fermenter was consisted of batch,feed-batch and induction phases.The relationship between dry cell weight(y) and culture time (t) in growth phase(batch and feed-batch phase)could be described by model y=0.6525e~(0.1907t).Glycerol and ammonia were almost used for cell growth and maintain,and no by-product was observed in batch and fed-batch phase Only 80% ammonia and 70% methanol were used by cell in induction phase.By comparison the results of shake-flask and 50L fermenter,it was concluded that the limit- ing factor in the fermentation of shake-flask and 50L fermenter was dissolved oxygen(DO)and.carbon source,respectively.When scaling the result of shake-flask to 501.fermenter,the control strategy was adapted for 50L fermenter by increasing the feed rate of methanol and the maximum PIP concentration reached 1.72 g/L.

Study on the Recombinant Human ApoA-I_Milano High Density and High Expression by Two Temperature-Shifted Induction in Escherichia coli / 微生物学通报

The temperature effect on the recombinant protein production formation was investigated in present study. The culture temperature of growth phase is 30℃, and the culture temperature of induction phase was arranged according to three modes. Hign cell-density and high expression culture of E.coli to product recombinant human apolipoprotein A-I Milano by two temperature-shifted induction . Two temperature-shifted induction was carried out high density and high expression recombinant human ApoA-1 Milano. The recombinant protein ApoA-I Milano reached 4.8 g?L -1 with the final cell density of OD 600 150. And the two temperature-shifted induction avoided the acetic acid successfully to the influence of the high density and high expression. Two temperature-shifted induction was viable in high density culture and high expression of heterogenous protein in recombination E.coli.The sduty provides a basic work for production of recombinant ApoA-I Milano in scale.

Optimation of the Fermentation Conditions for Human ApolipoproteinA-I Expression / 微生物学通报

Optimization of the fermentation condition for human apolipoproteinA-I expression in recombinant Escherichia coli was investigated. The recombinant plasmid pBV220-ApoA-I was transformed respectively into different E.coli hosts such as JM109, BL21(DE3),DH5?, BMH7118,and TG1. The best host E.coli was DH5? in which the recombinant ApoA-I expression percentage was 21.2% corresponding to that in BL21(DE3) in flask shaker cultivation,while the ApoA-I expressed percentage in E.coli TG1 was 11%.Fed-batch cultivation was performed in FMG-5L fermentor,the optimum fermentation cultivation conditions were as following :optimum pH value was 7.0 in growth phase and 7.4 in the expression phase. The initial glucose concentration in batch phase was 3 g?L -1.The optimum C/N ratio was 2∶1.The recombinant ApoA-I reached about 40% of the total protein, and concentration of ApoA-I was 2.86 g?L -1.