Efficient resolution of venlafaxine and mechanism study via X-ray crystallography.

Numbers of resolving factors were investigated to improve resolution of venlafaxine 1. An effective resolving agent, O,O'-di-p-toluoyl-(R, R)-tartaric acid 2, was screened using similar method of 'Dutch resolution' from tartaric acid derivatives. The resolution efficiency was up to 88.4%, when the ratio of rac-1 and 2 was 1:0.8 in THF with little water (10:1 v/v). Enantiomerically pure venlafaxine was prepared with 99.1% ee in 82.2% yield. The chiral resolution mechanism was first explained through X-ray crystallographic study. One diastereomeric salt with well solubility forms a columnar supramolecular structure as the acidic salt (R)-1·2, while the other diastereomeric salt with less solubility forms a multilayered sandwich supramolecular structure by enantio-differentiation self-assembly as the neutral salt 2(S)-1·2. The water molecules play a key role in the optical resolution, as indicated by the special structures of the diastereomeric salts.

Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by P(GAP)-driven Pichia pastoris overexpressing methionine adenosyltransferase.

Methionine adenosyltransferase (MAT) was overexpressed within Pichia pastoris employing the promoter of glyceraldehyde-3-phosphate dehydrogenase gene (P(GAP)), to biosynthesize S-adenosyl-l-methionine (SAM). Effects of five glycerol feeding tactics on MAT activity were first investigated. Strategies A-C were based on limited feeding correlated with dissolved oxygen (DO) at 50.0%, 25.0% and 0.0%, respectively. For strategies D and E, unlimited supplementation was executed by pulsed feeding mode. Gradual decline (2-0%) (w:v) of the residual glycerol level was shown between any two pulses in strategy D, while a nearly stable content (2%) throughout fed-batch cultivation with strategy E. With shifting strategies A-E in alphabetical order, gradual improvements of MAT activities were achieved, with the maximum of 9.05Ug(-1) dried biomass for strategy E, since the specific glycerol consumption rate (F(G)) ascended due to the elevated specific oxygen uptake rate (qO(2)). The success was ascribed to the enhancement of oxygen transfer rate (OTR), because 2% glycerol improved oxygen saturation content in broth (C*) and volumetric oxygen transfer coefficient (k(L)a). Strategy E also led to the highest values of ATP and biomass besides MAT. Consequently, the highest SAM yield and volumetric level were obtained at 0.058gg(-1) and 9.26gl(-1), respectively.

[Compared D-amino acid oxidase expression in different Pichia pastoris host strains].

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.

[Constitutive expression and purification of Alcaligenes faecalis penicillin G acylase in Escherichia coli].

Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions, the gene of PGA was cloned into an expressing vector pKKFPGA. The recombinant plasmid contained multicopy replicon(COLE 1), trc promoter, AfPGA gene, rrnB transcript terminator and ampicillin marker transformed Escherichia coli DH5alpha. As both the recombinant plasmid and the host DH5alpha had no laclq gene, the trc promoter was always active and the AfPGA could be constitutively expressed without IPTG induction in the host DH5alpha. In the shaking flask, the recombinant cell was inoculated into the fermentation medium (tryptone 10g/L, yeast extract 5g/L, MgSO4 x 7 H2O 1g, KH2 PO4 2g/L, K2HPO4 x 3H2O 5g/L, Na2HPO4 x 12H2O 7g/L, (NH4)2SO4 1.2g/L, NH4Cl 0.2 g/L, NaCl 0.1g/L, dextrin 30g/L) and cultured at 28 degrees C for 20h. The production of AfPGA reached 2,590u/L(NIPAB method), with a cell-density-specific activity of more than 300(u/L)/A600, this yield increased 432 fold higher than the native expression of Alcaligenes faecalis . Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effluent was added ammonium sulphate to 1mol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-1mol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6u/mg protein. However, the overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of various types of PGA precursors and then formed inclusion bodies in the cytoplasm and/or periplasm. In this study, 5% PGA precursors formed as inclusion bodies in the cytoplasm while no inclusion bodies formed in the periplasm. It suggested most PGA precursors were transported to the periplasm and matured to active PGA and also explained why PGA gene was highly expressed in the host DH5alpha. On the other hand, inclusion bodies in the cytoplasm indicated that the maturation of PGA in the host DHSalpha was limited by the translocation step.

Improving the specific synthetic activity of a penicillin g acylase using DNA family shuffling.

Penicillin G Acylas (PGA) of Providencia rettgeri (ATCC 25599) was evolved using a modified DNA family shuffling method. The identity of pga genes from Escherichia coli, Kluyvera citrophila and Providencia rettgeri ranges from 62.5% to 96.9%. The pga genes from above three species were recombined and shuffled to create interspecies pga gene fusion libraries. By substituting assembled chimaeras for corresponding region of pETPPGA, different recombinants were constructed and expressed in E. coli JM109(DE3). Mutants with obvious beta-lactam synthetic activity were selected from the plates and the ratios of synthesis to hydrolysis (S/H) were determined subsequently. It was shown that the primary structures of selected positives exhibited significant diversity among each library. The best mutant possessed 40% higher synthetic activity than the wild type enzyme of PrPGA. It was further proved in this study that the domain of alpha subunit contributed much more to improve the specific activity of synthesis. Results showed a recombinant PGA with higher synthetic activity was acquired by the method of DNA shuffling.

Purification and characterization of Alcaligenes faecalis penicillin G acylase expressed in Bacillus subtilis.

The Alcaligenes faecalis PGA gene encoding heterodimeric penicillin G acylase (PGA) was cloned and successfully expressed in Escherichia coli and Bacillus subtilis respectively. In contrast to E.coli hosts where the enzymes were retained in the periplasm, B. subtilis cell secreted the recombinant enzyme into the medium. Contrary to limited expression yield of E. coli (pETAPGA), PGA extracellularly expressed by B. subtilis (pBAPGA) and B. subtilis (pMAPGA) reached the highest yield of 653 u/L. This yield increased 109-fold higher than the native expression of A. faecalis CICC AS1.767. The enzyme was fractionated with (NH(4))(2)SO(4) and purified by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific activity of 1.469 u/mg. Furthermore, some enzyme characteristics, such as the pH and temperature optimum, the stability against organic solvent and the ratio of cepholexin synthesis to hydrolysis were determined. By overexpressing A. faecalis PGA in B. subtilis and purifying secreted enzyme from culture medium one could readily obtain a large amount of an alternative source of PGA.

[Enhancement of the production of SAM by overexpression of SAM synthetase in Pichia pastoris].

S-Adenosyl-L-methionine(SAM) is an important metabolic intermediate in the metabolic flux of sulphur. SAM is involved in three key metabolic pathways: transmethylation, transsulfuration and polyamine synthesis. As a potential therapeutic agent, SAM is being used as over the counter drug and nutrient supplement. An expression vector, harboring SAM synthetase 2 gene from S. cerevisiae and regulated by the glyceraldehyde-3-phosphate dehydrogenase gene promoter P(GAP), was transformed into GS115 strain of P. pastoris. Through zeocin resistance and expression screening, a recombinant strain was obtained that had high SAM yield and the fermentation conditions were optimized. The results showed that carbon source, nitrogen source, pH and dissolved oxygen had significant effects on the accumulation of SAM. The SAM production of the recombinant cells reached 2.49 g/L after fermentation for three days under the optimized conditions. The present studies show that fermentation of recombinant P. pastoris strain, expressing heterologous SAM synthetase gene, may be a promising approach for the production of SAM.

[The pH-dependent catalytic reaction of penicillin G acylase and its mutants].

The pH-dependence in the catalytic reaction of recombinant penicillin G acylase and its mutants from B.megaterium has been studied by using kinetic methods. pK(1) and pK(2)of the residues of the wild type penicillin G a cylase, involved in the catalyzed reaction, were 5.50-5.87 and 10.73, respectively, from the curves of logV(m) and log(V(m)/K(m)) versus pH. Results showed tha t the pK(1) and pK(2) values of these residues of the mutants were similar to that of the wild type. pK(1) of 5.64-5.86 for mutant A and 5.69-6.96 for mutant B were obtained, while pK(2) was 10.61 and 10.48 for mutant A and B, respectively. At the same time, pK values at different temperatures were investigated. The ionization enthalpies(deltaH) were 44.38-59.03 kJ/mol and 147.37 kJ/mol, respectively, from th e curve of pK versus temperature. It was presumed according to the results mentioned above that the ionizing residues, involved in the reaction, wer e histidine and lysine that are localized around the active site.

[Production of SAM by recombinant Pichia pastoris].

To utilize Pichia pastoris to produce S-adenosyl-L-methionine (SAM), an intracellular expression vector harboring S. cerevisiae SAM2 was transformed into GS115. A recombinant strain having 2 copies of expression cassette was obtained through G418 resistance screening. This strain had higher SAM synthetase activity and higher SAM production capacity than the original strain, when cultured in medium containing methanol and methionine. The carbon source and nitrogen source of medium was optimized. The results showed SAM production by this strain was closely related to carbon metabolism. With supplementation of 0.2% glycerol every day from the beginning of 3rd day, this strain produced 1.58 g/L SAM when cultured in a medium containing 0.75% L-methionine and optimized carbon and nitrogen source after 6 days.

PEG and DBBF modified porcine hemoglobin and their oxygen-carrying capacity.

Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge molecule size of proteins, to prolong their retention time in the circulation as well as blunt immune or allergic reactions. Hemoglobin cross-linked with small molecular modifiers turns out to be more stable and to have better oxygen carrying capacity. In the present study, four derivatives of PEG with different activation groups, and several PEGs with different molecular weights were covalently bound to porcine hemoglobin (pHb). PEG-pHbs exhibited a variety of differences in their properties depending on the molecular weights of the used PEGs, the amounts of bound PEGs and the presence or absence of allosteric cofactors. The optimal modification conditions for bis (3, 5-dibromosalicyl) fumarate (DBBF) as well as the physical features and oxygen carrying capacity of DBBF-modified pHb were evaluated. Furthermore, both PEG and DBBF were used simultaneously to modify pHb. The results indicate that the pHbs modified with PEG and DBBF have more stable tetrameric conformations with a molecular weight of 107 kD. Their oxygen half-saturation pressure (P(50)) is around 3.33 kPa which approximates the physiological P(50) of human red blood cells.

Enhancing Penicillin G Acylase Stability by Site-directed Mutagenesis.

To improve the stability of penicillin G acylase(PGA) from Bacillus megaterium, a three-dimensional model of B.megaterium PGA was constructed based on crystal structure of penicillin G acylase from E.coli using PMODELING program. The mutation of Lys at beta427 and 430 to Ala was predicted to enhance the stability of PGA in acidic or organic solvent environment. The results showed that 2 mutant PGA had similar specific activity and Km as the parent PGA. Their optimum pH dropped 0.5 pH units. The stability of Lys430Ala was enhanced obviously at pH 5.2. The half lives of Lys427Ala and Lys430Ala were improved by 60 % and 166 %, respectively, in comparison with the parent PGA.

Preparation of Inositol Tetrakisphosphate and Its Application in Modification of Porcine Hemoglobin.

Modification of hemoglobin using inositol tetrakisphosphate (IP(4)) can improve the oxygen affinity of hemoglubin. Phytase was extracted from wheat bran and purified by ammonium sulphate fractionation, followed by Sephadex G-50 gel filtration and Mono Q chromatography. The purified phytase was used for hydrolysis of phytic acid under controled conditions. IP(4), as a major composition of the hydrolysate, was further purified on resin 714 column. The purified IP(4) was oxidized by periodate to obtain dialdehyde-IP(4). By the reaction between the aldehyde group of IP(4) derivative and the amino group of porcine hemoglobin (pHb), pHb-IP(4) conjugate was formed and was found to have better ability of O(2) binding and releasing than the native hemoglobin.

High Expression and Purification of Recombinant Human Serum Albumin from Pichia pastoris.

Recombinant human serum albumin (rHSA) was produced in methylotrophic yeast Pichia pastoris. By optimization of expression, about 150 mg/L of rHSA was obtained from broth of Pichia pastoris GS115/HAS (his+Mut(S)) supernatant. The rHSA was isolated and purified by hollow-fiber ultrafiltration, Phenyl-Sepharose hydrophobic chromatography and antibody-immunoadsorbent chromatography. Finally, rHSA was purified to electrophoretic purity.

High Expression of Penicillin G Acylase Gene from Bacillus megaterium in Bacillus subtilis.

The penicillin G acylase gene (pga) amplified by PCR from Bacillus megaterium was subcloned into an expressing vector pPZW103 (P43 as promoter). The recombinant plasmid was transferred into Bacillus subtilis DB104. Penicillin G acylase production in the B. subtilis transformant was 3-6 u/ml, higher than that of published recombinant strains. Penicillin G acylase production was induced by phenylacetic acid in B. megaterium, whereas the enzyme was produced constitutively in the B. subtilis transformant carrying B. megaterium pga. The recombinant strain showed high stability in antibiotic-free medium for 10 days. Enzyme in crude broth was purified by Al(2)O(3) chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography and the total yield is 79%. The purified enzyme with specific activity of 52 u/mg can be directly immobilized for use.

Oxygen-binding Properties of Complexes between Dextran and Pyridoxal 5-Phosphate-Modified Porcine Hemoglobin.

It was demonstrated by oxygen equilibrium curve that the pyridoxal 5-phosphate(PLP) modified porcine deoxyhemoglobin(pHbbeta) had lower oxygen affinity than that of stroma-free porcine hemoglobin(pHb). Accourding to analysis of SDS-PAGE, the porcine hemoglobin derivatives were not cross-linked between its subunits. A conjugate was synthesized between pHbbeta and p-toluenesolfonyl chloride-actived dextran. The oxygen affinity of Dx-pHbbeta was high than that of pHbbeta, but still lower than that of pHb. Judged by cellulose acetate film electrophoresis, the mobility of Dx-pHbbeta was apparently different from that of pHbbeta. Dx-pHbbeta has characterized absorbance peak in UV spectrum, which can be used to analysis the binding ratio between Dx and pHbbeta.

Oxidized Raffinose Intramolecular Cross-linking of Porcine Hemoglobin Effects on Its Structure and Function.

Periodate-oxidized raffinose is utilized to modify porcine oxyhemoglobin and deoxyhemoglobin, respectively. These modified protein derivatives showed special characteristics that it can resist the dissociation of alpha(2)beta(2) tetramer in comparison with natural proteins. According to the analysis in SDS-PAGE and reverse phase HPLC, the two porcine hemoglobin derivatives are all intramolecular linked proteins and the cross-linking sites lie between the two beta subunits. No eminent differences were found in the UV-VIS absorption spectrum between natural and modified proteins. However, after calibration for protein concentration, the emission of modified porcine deoxyhemoglobin is much weaker than the modified oxyhemoglobin and the natural protein. Furthermore, the porcine hemoglobin derivatives' characteristics of the oxygen affinity and Auto-oxidized rate also show significant differences with natural protein. The possible application of these porcine hemoglobin derivatives in the preparation of hemoglobin-based blood substitutes is discussed.

A Novel Reaction Media for Horseradish Peroxidase (HRP) Catalysis in Organic Solvents.

It is important to select favorable reaction media for use of enzymes in organic synthesis. In the presence of PEG with a certain molecular weight, toluene was able to dissolve horseradish peroxidase and the latter maintained a high activity. The HRP reaction conditions, such as ratio of PEG/toluene, water content, pH, and substrate concentration were investigated. It was found that the activity of HRP increased when PEG concentration was low and water content was high. In such a system, the enzyme functioned best at pH 7.0,the concentration of H(2)O(2) and guaiacol being 20 mmol/L and 0.1 mol/L respectively. Under the same condition, no activity was detected in the PEG / ethanol and PEG / dioxane systems. However, in PEG / toluene system, HRP maintained a higher activity and the activity was ten-fold more than that in toluene alone.

PEG-Modified Horseradish Peroxidase and Its Properties in Nonaqueous Media.

Modification of enzymes can enhance their activities in organic solvents. Horseradish peroxidase (HRP) was modified with methoxy-PEG 5000 which was linked onto HRP peptidal free aminos or onto the aminos introduced through the oxidation of sugar side chains of HRP. It was found the enzyme with PEG linked to peptide chain expressed a nearly same activity as the native HRP in aqueous system. However, PEG modified HRP by oxidation of carbohydrate chains remained only about one third activity of the native HRP. The same results were observed in reversed micelles and in dioxane of less than 30%. Apparently, both PEG modified HRP had the higher activities in high concentration of dioxane. Especially, the activities of the modified HRP in toluene were both enhanced up to nearly 3 fold. When HRP was modified with PEG through the amino groups of peptide chain, it was more stable than the native enzyme in water, dioxane and toluene. However, the stability of PEG-modified HRP by carbohydrate chains was higher in water and lower in both dioxane and toluene in comparison with native HRP.

Cloning and Expression of the pga Gene of Bacillus megaterium in E. coli.

The pga gene was amplified by PCR from Bacillus megaterium CA4098, cloned into pKK223-3 vector and expressed in E. coli HB101. The pga sequence was also determined. The deduced amino acids sequence was compared with those of PGAs from other bacteria and showed that they were similar, especially the active sites were strongly conserved.

The Effect of Reversed Micelles on Horseradish Peroxidase (HRP) Structure and Function.

Effects of water content (W(0)) and surfactants on the activity of HRP in CTAB/isooctane-n-pentanol reversed micelles were investigated. Based on UV-Vis spectroanalysis and the activity of HRP at various W(0) and CTAB content, it was found that the active-site of HRP was mainly influenced by reversed micelles. However, CTAB had no effect on the active-site of HRP. In addition, the stabilities and Soret band of HRP-H(2)O(2) complexes in reversed micelles were compared to that in aqueous system. The complex HRP-II and HRP-III did disappear more rapidly in reversed micelles than in aqueous system. This demonstrated the enzyme molecules were destabilized in reversed micelles.