Chul Soo Shin

Enhanced Production of Human Mini-Proinsulin in Fed-Batch Cultures at High Cell Density of Escherichia coli BL21(DE3) [pET-3aT2M2]

Synthesis of recombinant protein (human mini‐proinsulin) is investigated in fed‐batch cultures at high cell concentration of recombinant Escherichia coli BL21(DE3) [pET‐3aT2M2]. Transcription of the recombinant gene is controlled by a T7 promoter system. The human mini‐proinsulin is characterized by a C‐chain peptide consisting of only nine amino acids, whereas the C‐chain peptide of natural human proinsulin is made up of 35 amino acids. It is expressed in a fusion protein with a small fusion partner (a peptide with 18 amino acids) and finally aggregated into insoluble inclusion bodies in cytoplasm of recombinant E. coli. The fermentative production of this small fusion mini‐proinsulin may be of great advantage in enhancing the yield of human insulin. To find an optimum induction strategy, effects of various key cultivation variables on the mini‐proinsulin production are examined in high cell density fed‐batch cultures. No general correlation is found between preinduction specific growth rate and recombinant protein synthesis, which confers a flexibility in choosing the feeding strategy of preinduction media for achieving the high cell density cultures. A culture temperature below 37 °C is unfavorable for recombinant gene expression, and the T7‐based expression system is almost completely repressed at 30 °C. The nutrient glucose and yeast extract concentration in postinduction feed media is optimized by applying a statistical method for medium optimization, i.e. response surface methodology, and an effective amount of inducer molecule (IPTG) is determined to maximize the specific recombinant protein formation. The mini‐proinsulin production in E. coli culture is significantly influenced by the volumetric feed rate of postinduction media, which is shown to be closely related to the plasmid copy number in the recombinant cell. Consequently, in a single‐stage fed‐batch process, the mini‐proinsulin concentration is increased up to 7 g/L, approximately 62 wt % of which corresponds to mature human insulin. A two‐stage fed‐batch fermentation process, with recombinant cell growth occurring at a constant growth rate and constant cell concentration in a growth fermenter and mini‐proinsulin production in an induction fermenter, is designed, and its efficacy in increasing volumetric productivity of mini‐proinsulin is demonstrated.

Morphological change and enhanced pigment production of Monascus when cocultured with Saccharomyces cerevisiae or Aspergillus oryzae

When a Monascus isolate, a producer of Monascus pigments, was cocultured with either Saccharomyces cerevisiae or Aspergillus oryzae in a solid sucrose medium, there were significant morphological changes in Monascus culture. Cocultures exhibited cell mass increases of 2 times and pigment yield increases of 30 to 40 times compared to monocultures of Monascus. However, enhanced cell growth, an increase in pigment production, and morphological change did not occur in coculture with Bacillus cereus. Saccharomyces cerevisiae was more effective at enhancing pigment production than Asp. oryzae. Enhanced cell growth and increased pigment production occurred only in conjunction with morphological changes. Culture filtrates of S. cerevisiae were also effective in inducing morphology change in Monascus, similar to culture broths of S. cerevisiae. The hydrolytic enzymes produced by S. cerevisiae, such as amylase, and chitinase, are thought to be the effectors. The commercial enzymes alpha-amylase and protease from Asp. oryzae both caused a morphological change in Monascus and were effective in enhancing pigment production. However, lysozyme, alpha-amylase and protease from Bacillus species, protease from Staphylococcus, and chitinase from Streptomyces were not effective. The hydrolytic enzymes which cause a morphological change of Monascus culture and enhancement of pigment production are thought to be capable of degrading Monascus cell walls. An approximate 10-fold increase in pigment production was observed in liquid cocultures with S. cerevisiae. Copyright 1998 John Wiley & Sons, Inc.

High-level production of human growth hormone in Escherichia coli by a simple recombinant process

Procedures have been devised for producing in Escherichia coli high yields of purified recombinant human growth hormone (hGH), by utilizing N-terminal pentapeptide sequence of human tumor necrosis factor-alpha, histidine tag and enterokinase cleavage site as a fusion partner. The fusion protein was produced as a soluble protein at the beginning of gene expression, but progressively became insoluble in Escherichia coli cytoplasm. The insoluble protein was solubilized by simple alkaline pH shift and purified to near homogeneity by Ni(2+)-chelated affinity chromatography. Following specific enterokinase cleavage, the recombinant hGH was purified by one-step anion exchange chromatography. The ease and speed of this recombinant process, as well as the high productivity, makes it adaptable to the large-scale production of hGH. Moreover, the highly efficient fusion partner could be applied to the production of other therapeutically important proteins.

Morphology control of Monascus cells and scale-up of pigment fermentation

The morphology of Monascus cells was controlled and pigment fermentations scaled up from a 5 l jar to a 300 l pilot vessel. At a rotational speed of 350 rpm or less, serious mycelial aggregation was present with pigment yields of approximately 37.5 OD units. Monascus cells with long hairy mycelia were induced, resulting in viscous cultures. As the rotational speed increased from 350 to 700 rpm, the kLa values increased gradually from 0.003 to 0.029 s−1. A maximum pigment yield of 220 OD units was obtained at 500 rpm where mycelia with short branches were induced. The specific oxygen uptake rates (qO2) at 600 and 700 rpm were also lower than the rate at 500 rpm. Mycelia had short branches and appeared to be damaged by mechanical shear forces due to high agitation speeds. When seed cultures were grown in jar fermenters under high shear stress, the pigment yields and DOT values of fermentations were also high. Fermentations were successfully scaled up from 5 l jars to a 300 l pilot vessel based on a constant impeller speed of 12,500 cm min−1, which was the best condition for pigment production and cell morphology.

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone l−1 and 7 g fusion glucagon l−1. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content.

Polyphenol Compounds and Anti-inflammatory Activities of Korean Black Raspberry (Rubus coreanus Miquel) Wines Produced from Juice Supplemented with Pulp and Seed

Three types of Korean black raspberry wine were produced via alcoholic fermentation from juice, juice-pulp, and juice-pulp-seed, respectively. These wines were compared in terms of their anti-inflammatory activities and polyphenol contents. The total content of polyphenol compounds in wines was increased by 22.4% after supplementation with pulp and by 56.7% after supplementation with both pulp and seed. The reduction rate of NO evolution was highest in the order juice-pulp-seed wine, juice-pulp wine, and juice wine. Addition of the juice-pulp-seed wine at a level of 62.5-500 mg/L decreased the NO evolution rate by 40.5-94.2%. Eight fractions were obtained from juice-pulp-seed wine via ethyl acetate extraction and silica gel chromatography. Of these, the AF fraction, which exhibited the highest in vitro anti-inflammatory activity, exerted inhibitory effects on ear edema, writhing response, and vein membrane vascular permeability in mice. 3,4-Dihydroxybenzoic acid accounted for 37.6% of the total polyphenol content in the AF fraction.

Continuous l-cysteine production using immobilized cell reactors and product extractors

Abstract Methods to improve the stability of l -cysteine-producing enzymes from Pseudomonas sp. M-38, both as whole cells and as immobilized cells, were investigated for the production of l -cysteine from d,l -2-amino-Δ 2 -thiazoline-4-carboxylic acid ( d,l -ATC). Among the three l -cysteine-producing enzymes only l -ATC hydrolase was unstable. However, the stability of l -ATC hydrolase was significantly enhanced by the addition of 20% sorbitol. In continuous l -cysteine production, more than 60% of the initial activity of l -ATC hydrolase remained after 1000 hours at 37°C with 40% sorbitol and at 30°C with 20% sorbitol. A system involving a cascade of processes using two packed-bed reactors with immobilized cells and two l -cysteine extractors with the ion-exchange resin Dowex 50W was developed to reduce product inhibition and unreacted substrate. The overall productivity of the system was 43% higher than for two reactors without an ion-exchange extractor.

Imprinted polymers as tools for the recovery of secondary metabolites produced by fermentation

Imprinted polymers were synthesized using a mixture of pigments, N-glutamyl-rubropuctamine, and N-glutamyl-monascorubramine (I) as the template, and 2-methacrylamido-6-picoline or 4-aminostyrene as functional monomers, to obtain recognition materials capable of forming hydrogen bonds and charge interactions, respectively, with carboxyl groups of target I in the binding sites. The polymers were prepared thermally at a template loading of 5 mol% using ethylene-glycol dimethacrylate or trimethylolpropane trimethacrylate as crosslinkers and acetonitrile or tetrahydrofuran as porogens. The selective binding of I to both types of polymer was demonstrated, although aminostyrene-based materials showed higher overall adsorption and were studied in more detail. It was shown that the kinetics of binding of I from ethyl-acetate extracts of fermented Monascus sp. was very rapid and virtually all the pigment adsorbed can be released by washing the polymer with ethanol-water mixtures. The feasibility of reusing imprinted polymer in consecutive adsorption/desorption cycles was also demonstrated. Copyright 1999 John Wiley & Sons, Inc.

Reduced formation of byproduct component C in acarbose fermentation by Actinoplanes sp. CKD485-16

Acarbose fermentation was conducted by cultivation of Actinoplanessp. CKD485–16. Approximately 2,300 mg/L of acarbose was produced at the end of cultivation along with 600 mg/L of the acarbose byproduct component C. Maltose, a known moiety of acarbose, should be maintained at high concentration levels in culture broths for efficient acarbose production. The acarbose yield increased with an increasing osmolality of the culture medium, with a maximum value of 3,200 mg/L obtained at 500 mOsm/kg. Component C was also produced in proportion to the osmolality. Conversion of acarbose to component C was accomplished with resting whole cells. Inhibitors of the conversion of acarbose to component C were sought since component C is probably derived from acarbose. Valienamine was found to be a potent inhibitor, resulting in a more than 90% reduction in component C formation at a 10 μM concentration. Effects were similar in a 1,500‐L pilot fermentor with acarbose and component C yields of 3,490 and 43 mg/L at 500 mOsm/kg, respectively.

Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

SummaryA mutant strain lacking in activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was screened after UV-treatment ofPseudomonas sp. CU6. The properties of the two strains, original and mutant, were compared on the basis of parameter values estimated from kinetic simulations of the enzymatic formation of L-cysteine from D,L-ATC. Both strains suffered from product inhibition, though inhibition was less for the mutant strain.