l-Serine synthesis using the resting Hyphomicrobium sp. NCIB10099 cells under suppressive conditions for l-serine-degrading activity

l-Serine-degrading activity could be suppressed by controlling the methanol concentration in l-serine synthesis using resting Hyphomicrobium sp. NCIB10099 cells. Fifty-three mg/ml of l-serine were produced from 100 mg/ml of glycine and 104 mg/ml of methanol. It is believed that l-serine-degrading activity contributes to the reverse serine hydroxymethyltransferase reaction.     

New bacteriophages active on strains of Hyphomicrobium

Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (Tm) ranged from 67 to 93 degrees C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.     

l-Serine production by a methylotroph and its related enzymes

The production process of l-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of l-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32–34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers. Correspondence to: Y. Izumi     

Molecular characterization of dichloromethane-degrading Hyphomicrobium strains using 16S rDNA and DCM dehalogenase gene sequences

A phylogenetic analysis of 6 strains of dichloromethane (DCM) utilizing bacteria was performed. Based on the almost complete 16S rDNA sequence determination, all strains clustered together and showed high sequence similarity to Hyphomicrobium denitrificans, except for the strain MC8b, which is only moderately related to them and probably represents a distinct species. The 16S rDNA-based phylogenetic tree was compared to the one obtained from the DNA sequence data of the dcmA gene coding DCM dehalogenase, the key enzyme of DCM utilization. The topology of the two trees is in good agreement and may suggest an ancient origin of DCM dehalogenase, but also raises questions about the original role of the enzyme.     

l-Serine Production by a Mutant of Sarcina albida Defective in l-Serine Degradation

For improved l-serine production, an l-serine dehydratase-defective mutant of Sarcina albida IAM 1012 was obtained. In the mutant, the activities of the enzymes responsible for l-serine production were as high as those in the parent strain, and, at a low glycine concentration, the mutant accumulated l-serine more efficiently than the parent. Under optimum conditions, 21 mg of l-serine per ml accumulated from 100 mg of glycine per ml. l-Serine was isolated from a reaction mixture as l-serine m-xylene-4-sulfonate, and free amino acid was obtained in high yields by use of an ion-exchange resin. Residual glycine was recovered at a yield of 61%.     

Secretory production system of bionanocapsules using a stably transfected insect cell line

Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host–vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 μg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.     

Hyphomicrobium as a component of the aquatic microflora--morphological and physiological studies on two strains

Aspects of the morphology, metabolism and physiology of two oligocarbophilic strains of Hyphomicrobium (H4K and S5K), isolated from the Plusssee, were studied. Both strains are able to grow on mineral salts media without added organic carbon sources. Strain H4K grows well even in double distilled water. The two strains cannot grow on mineral media in the absence of atmospheric CO2. No growth occurred also in air purified of organic carbon, in spite of the presence of CO2. On the contrary, there is good growth in the presence of some organic compounds and without atmospheric CO2, i.e., heterotrophic metabolism without CO2 assimilation is possible. Growth was enhanced in a methanol atmosphere, and by the addition of yeast extract, methylamine, peptone and glucose. In nutrient solutions containing acetate or formate as carbon source, growth of H4K begins only after an adaptation period of ca. 4 weeks.     

Optimization of ascorbic acid-2-phosphate production from ascorbic acid using resting cell of Brevundimonas diminuta

With the aim to produce ascorbic acid-2-phosphate (AsA-2-P) from L-ascorbic acid (AsA, Vitamin C), nine bacteria conferring the ability to transform AsA to AsA-2-P were isolated from soil samples alongside known strains from culture collections. Most isolates were classified to the genus Brevundimonas by 16S phylogenetic analysis. Among them, Brevundimonas diminuta KACC 10306 was selected as the experimental strain because of its the highest productivity of AsA-2-P. The optimum set of conditions for the AsA-2-P production from AsA using resting cells as the source of the enzyme was also investigated. The optimum cultivation time was 16 h and the cell concentration was 120 g/l (wet weight). The optimum concentrations of AsA and pyrophosphate were 550 mM and 450 mM, respectively. The most effective buffer was 50 mM sodium formate. The optimum pH was 4.5 and temperature was 40 degrees C. Under the above conditions, 27.5 g/l of AsA-2-P was produced from AsA after 36 h of incubation, which corresponded to a 19.7% conversion efficiency based on the initial concentration of AsA.     

Evidence for the existence of isocitrate lyase activity in methylotrophic Hyphomicrobium strains

Abstract Isocitrate lyase activities were detected in a range of 0.096–0.212 units mg⁻¹ in cell-free extracts of all tested Hyphomicrobium strains grown on methanol as a sole carbon source, although the activities were rapidly lost during storage at 4 °C. When cell-free extracts were incubated with dithiothreitol, after storage the recovery of activity was observed, indicating the involvement of a labile sulfhydryl group in the enzyme. This confirmed the distribution of unstable isocitrate lyase in the genus Hyphomicrobium , and, contrary to previous observations, the operation of the ic ⁺-serine pathway was suggested for the assimilation of one-carbon compounds.     

Microbial glycolipid production under nitrogen limitation and resting cell conditions.

Rhodococcus erythropolis is able to synthesize an anionic trehalose-2,2',3,4-tetraester during cultivation on n-alkanes. Preconditions for an overproduction are nitrogen limitation, temperature- and pH-shift. The optimum carbon source was technical grade n-C-10, which led to 0.35 g g-1 of glycolipid per n-alkane. Electron microscopical observations showed that n-C-14,15 (technical grade) grown cells contained numerous lipid inclusions in contrast to n-C-10 (technical grade) grown cells. Nocardia corynebacteroides synthesizes a novel pentasaccharide lipid and as size products small amounts of trehalose-corynomycolates. Optimum precursors for overproduction are n-alkanes from n-tetradecane to n-hexadecane with yields in the range of 0.17 g g-1 of glycolipid per carbon source.     

Protein production using the baculovirus‐insect cell expression system

The baculovirus‐insect cell expression system is widely used in producing recombinant proteins. This review is focused on the use of this expression system in developing bioprocesses for producing proteins of interest. The issues addressed include: the baculovirus biology and genetic manipulation to improve protein expression and quality; the suppression of proteolysis associated with the viral enzymes; the engineering of the insect cell lines for improved capability in glycosylation and folding of the expressed proteins; the impact of baculovirus on the host cell and its implications for protein production; the effects of the growth medium on metabolism of the host cell; the bioreactors and the associated operational aspects; and downstream processing of the product. All these factors strongly affect the production of recombinant proteins. The current state of knowledge is reviewed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:1–18, 2014     

Multi-criteria comparison of resting cell activities of bacterial strains selected for biodesulfurization of petroleum compounds

Five isolates able to utilize dibenzothiophene (DBT) as a sole sulfur source and to convert it to 2-hydroxybiphenyl (HBP) with high rates were selected to investigate their potentialities as biocatalysts of a diesel oil biodesulfurization process. Conventional and chemotaxonomic analyses and 16S ribosomal DNA (rDNA) sequencing showed that these strains belonged to the Rhodococcus/Gordonia cluster. The desulfurizing activities of resting cells were compared under various conditions to evaluate their stability in both aqueous and organic media, their sensitivity to the presence of hexadecane and their sulfur substrate selectivity. In spite of their taxonomic similarity, the five strains exhibited different properties. This diversity was not confirmed by the analysis of the desulfurizing genes by amplification and sequencing of large fragments of dszA, dszB, dszC, and dszD genes which revealed that four of the five selected strains had a dsz genotype identical to those of the reference strain, Rhodococcus erythropolis IGTS8.     

A two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system

A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 mug CAT/(10(6) cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high-density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high-density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high-density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two-stage bioreactor system was proven to be reliable for a long-term operation beyond 600 h. (c) 1993 John Wiley & Sons, Inc.     

Pilot plant conversion of steroid using resting cell suspension as biocatalyst

Resting cell suspensions of Sepedonium ampullosporum have been used successfully in the pilot plant, transformation of 9β 10α-pregna-4, 6-dien-3, 20-dione to its 16α-hydroxy derivative. The resting cell enzyme system acted as a stable respiratory unit up to 150 hr after resuspension in water. Semicontinuous addition of substrate to the same cell suspension reduced overall conversion lime by 67%. Aeration and agitation were important factors affecting conversion rates. The hydroxylating system had a critical oxygen concentration above 90% saturation of air in water. Hydroxylase activity was inhibited by cyanide an d totally inhibited by the respiratory inhibitor antimycin A at concentrations much less that that concentration required to block normal respiration.     

Rapid identification of biosurfactant-producing bacterial strains using a cell surface hydrophobicity technique

Rapid identification of biosurfactant-producing bacterial strains was achieved by assaying cell surface hydrophobicity which had a direct correlation with biosurfactant production by Serratia marcescens, Pseudomonas aeruginosa, Bacillus pumilus, B. laterosporus, Acineto- bacter calcoaceticus, Escherichia coli and Staphylococcus aureus. These properties namely, Hydrophobic Interaction Chromatography, Salt Aggregation Test, Bacterial Adherence To Hydrocarbon and adhesion to polystyrene by Replica Plate test, provide a simple means for identifying bacteria associated with the production of biosurfactants.     

Continuous ethanol production using cell recycle with a settler

Summary A system for a high-density culture of Zymomonas mobilis was tested using a reactor with cell recycle by means of a simple settler. Growth-limiting conditions were created by raising the temperature and lowering the amount of yeast-extract. In this case the biomass production decreased, while the specific ethanol productivity did not change markedly. Under these conditions we succeeded in creating a system with a productivity of 40.5 g.1^-1.h^-1. The choice for optimal design of the settler and optimal conditions has to be made by optimization taking into account economic and whole process considerations.     

High-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system

In order to develop an efficient process for large-scale production of recombinant protein, various factors were studied which affect the productivity of Sf-9 (Spodoptera frugiperda) insect cells when using the baculovirus expression system. It was shown that upon infection with the Bac-BRV6L recombinant baculovirus, the level per cell of VP6 (a bovine rotavirus nucleocapsid protein) would drop 10-fold when host cell density at the time of infection increased from 2 x 10(6) to 3 x 10(6) cells/mL. The decrease was found to be totally reversible by culture medium renewal after infection, even when cells were infected at the stationary phase. Recombinant protein production was 4-6 times higher using TNMFH medium supplemented with 10% fetal bovine serum (FBS) than in IPL/41 serum-free medium. Fine-tuning of infection parameters in a 4-L surface-aerated bioreactor resulted in the production of typically 350 mg/L of VP6 protein, representing more than 25% of total cell proteins.     

l-Serine Production Improved by Analogue-resistant Mutants of a Methanol-utilizing Bacterium

In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.     

Vacuum fermentation for ethanol production using strains of Zymomonas mobilis

Summary Using strains of Z.mobilis, a vacuum fermentation system has been evaluated. The system was designed with the fermentor at atmospheric pressure and an external vacuum vessel (50 mm Hg). Sequential operation of the vacuum vessel was under microprocessor control. The use of Z.mobilis together with the two-stage design of the vacuum system has been found to overcome the problems of oxygen addition and the possibility of contamination reported previously for vacuum fermentations with yeasts. The productivity of 85 g/1/h found in the continuous cell recycle experiments was similar to that reported previously for a strain of S.cerevisiae.     

Continuous production of high-content fructooligosaccharides by a complex cell system

A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module was employed to produce high-content fructooligosaccharides (FOS) in a continuous process initiated by a batch process. The system used mycelia of Aspergillus japonicus CCRC 93007 or Aureobasidium pullulans ATCC 9348 with beta-fructofuranosidase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity. Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate. Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high-content FOS was discharged continuously from a MF module. The complex cell system was run at 30 degrees C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5-7% glucose and 8-10% sucrose on a dry weight basis, plus a small amount of calcium gluconate. The system worked for a 7-day continuous production process with a dilution rate of 0.04 h(-1), and the volumetric productivity for total FOS was more than 160 g L(-1) h(-1).