Characteristics of batch culture of a recombinant bacillus brevis excreting a foreign esterase

The productivity of extracellular enzyme was evaluated in batch culture using a protein hyperexcreting host, Bacillus brevis HPD31(5) harboring pHSC131, which carried a gene (est) encoding esterase activity from Bacillus stearother mophilus. Optimum temperature and pH for the bacterial growth and the production of extracellular esterase were found to be 35 degrees C and pH 6.5, by using the standard medium (GPY) containing neomycin as a selective pressure, Under the cultivation condition employed, cell growth reached 5 g dry cell weight/L, while the extracellular esterase activity amounted to 4.5 U/mL. Most (79%-92%) of the esterase produced was excreted into the medium. pHSC131 was stably retained in the host cell during cultivation in the presence of neomycin. However, in the absence of neomycin, the plasmid was completely lost from the host after 12-h cultivation accompanied by decreases in both esterase activity and production of total extracellular protein. The copy number of the plasmid was estimated to be approximately 7 throughout the cultivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the excreted proteins showed the presence of a protein having an apparent molecular weight of 32,000, which equals to the value predicted from the DNA sequence of the est gene.     

Coimmobilization of gluconolactonase with glucose oxidase for improvement in kinetic property of enzymatically induced volume collapse in ionic gels

The object of this paper is to provide an enzymatic means to attain faster swelling or shrinking kinetics of polyelectrolyte gels that undergo volume phase transition as an immobilized enzyme reaction sets in. For this, we studied the coimmobilization of gluconolactonase (GL) with glucose oxidase (GOD). A gel used was in the shape of a small cylinder (several hundred micrometers in diameter) and composed of a lightly cross-linked copolymer of N-isopropylacrylamide and acrylic acid. GL was isolated from Aspergillus niger and purified about 100-fold. It was found that in a substrate solution containing glucose, the gel with the coimmobilized GL and GOD shrinks very rapidly. The shrinking rate was identical to that of the enzyme-free gel that undergoes a shrinking transition in response to a sudden pH change of the outer medium from 7 to 5. This indicates the rate-limiting step in the shrinking process to be diffusion of the networks, but not the enzyme reaction. In the gel with singly immobilized GOD, a very slow shrinking was observed because the process is governed by the enzyme reaction. These results were discussed in full in connection with an enzymatically induced decrease in pH within and in the vicinity of the gel phase. As a result, it has become apparent that the faster shrinking kinetics in the coimmobilized enzyme system is attained by the GL-catalyzed hydrolysis of D-glucono-delta-lactone resulting from the oxidation of glucose with GOD.     

Optimized extracellular production of alkaline phosphatase by lky mutants of Escherichia coli K12

Summary Periplasmic-leaky (lky) Hfr mutant strains of Escherichia coli K12, grown in low-phosphate Tris medium, excreted alkaline phosphatase (AP) into the extracellular fluid. The lky207 mutation, which proved to induce the highest AP excretion rate, was transferred to an F^- host, carrying a phoS, T mutation allowing constitutive AP biosynthesis. Use of high-phosphate LB-rich medium for growing this F^- lky strain improved cell biomass, extracellular AP activity and excretion specificity in favour of the enzyme. Physiological studies helped us to develop a new culture medium (LB 8.3) giving higher enzyme and excretion yields. LB 8.3 medium also increased cell viability of lky mutants stored at 4° C.Using optimized culture conditions, the highest extracellular enzyme activity produced by lky mutant 706 was reached in the late stationary growth phase and was equal to 1,400 U/ml of culture medium (i.e., 6 times the intracellular AP content of wild-type strain, Ga15, developed in derepressed conditions); AP released into the extracellular fluid corresponded to 34% of total excreted proteins and was equivalent to a purified enzyme preparation.     

Metabolism characteristics of <Emphasis Type="Italic">Chelativorans oligotrophicus</Emphasis> by two-phase growth on the mixture of EDTA and glucose

The obligate destructor of ethylene diamine tetraacetate—a culture of Chelativorans oligotrophicus LPM-4—did not grow on a medium with glucose, but it was good to use it under cultivation on a mixture with EDTA after considerable decrease of the EDTA concentration in the medium (two-phase growth). Strong inhibition of hexokinase and glucose 6-phosphate dehydrogenase in cell exracts 4 mM EDTA was revealed. Using EDTA, cells accumulated polyphosphates whose rate decreased during glucose utilization phase. High activities of polyphosphate biosynthesis ferments (adenylat kinase and polyphosphate kinase) were distinguished during the first phase of the cultivation; considerable decrease of them and increase of polyphosphate glucokinase were found during the second phase of the cultivation. This points to the possible participating of polyphosphates in glucose metabolism as a supplementary energy source.     

Cellulase production by Trichoderma reesei

Summary A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h^–1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h^–1 all of these properties change due to derepression (for D<0.025 h^–1) or repression (for D>0.025 h^–1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.In a two-stage system the time to attain a steady state increases to 4–6 weeks. At low dilution rates the enzyme activity is only slightly higher in the second stage than in the first. At high dilution rates, at which the enzyme is not excreted into the medium in the first stage, enzyme activity can be increased considerably in the second stage.     

Fed-batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60-L working volume airlift tower loop reactor

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60-L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony-forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl-thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria-Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity. (c) 1993 John Wiley & Sons, Inc.     

The adaptation ofCandida utilis to dense recycled molasses mashes under continuous cultivation

A two-phase metabolism ofCandida utilis occurred during batch cultivation in a molasses mash. It was characterized by intensive accumulation of biomass without a lag, utilization of glucose, formation of acetate and ethanol and their conversion to ethyl acetate during the first phase. In the second phase the accumulation of biomass continued and was accompanied by simultaneous utilization of ethyl acetate or amino acids, contained in molasses or produced by the culture during the first phase. A content of betaine, ash, non-assimilable nitrogen and reducing compounds as well as osmotic pressure increased with increasing density in separated mashes. The culture adapted to this medium during a two-stage continuous cultivation divided according to the two-phase nature of the metabolism. In the course of the adaptation the culture developed the ability to utilize succinate, glutamate, citrate and other originally non-assimilable compounds. A specific growth rate and productivity of the system increased proportionally with the increased concentration of assimilable substrates during a transition from one steady state to another. The adaptation in batch culture was not successful.     

A novel acid phosphatase excreted by Penicillium funiculosum that hydrolyzes both phosphodiesters and phosphomonoesters with aryl leaving groups

An acid phosphatase has been purified from the culture broth of Penicillium funiculosum by procedures including SP-Sephadex column chromatography and Sephacryl S-200 gel filtration. The phosphatase appears to be a 76 kDa heterodimer composed of 51 and 26 kDa subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes both phosphodiesters and phosphomonoesters, but only those with aryl leaving groups. At the early phase of degradation of bis-p-nitrophenyl phosphate by the enzyme, inorganic phosphate and the intermediary product, p-nitrophenyl phosphate, are liberated at approximately the same rate. This indicates that the intermediary phosphomonoester produced on the enzyme is further hydrolyzed in situ or dissociates into the medium at approximately the same probability.     

Inorganic polyphosphates and phosphohydrolases from Halobacterium salinarium

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 mumol Pi/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content of H. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities of H. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase of H. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective Km of this enzyme with respect to pyrophosphate was 115 microM.     

The temporal regulation of protein synthesis during synchronous bud or mycelium formation in the dimorphic yeast Candida albicans

When stationary phase cells of the dimorphic yeast Candida albicans are diluted into fresh medium at 37°C at either pH 4.5 or pH 6.5, they evaginate at exactly the same time and with the same synchrony. However, they then grow in the budding yeast form at the former pH and in the elongate mycelium form at the latter pH. Three phases of protein synthesis are distinguished for cells forming either buds or mycelia: an initial 50-min period (phase I) during which total cell protein remains constant and the rate of incorporation of labeled amino acid into protein is virtually zero; a second period (phase II) during which there is a slow but constant increase in both total cell protein and the rate of incorporation; and a third period (phase III) during which there is a dramatic increase in both total cell protein and the rate of incorporation. The transition from phase I to phase II occurs at the same time for cells forming either buds or mycelia, but the transition from phase II to phase III occurs 20 to 30 min later in the mycelium than in the bud forming population, the same temporal difference observed for phenotypic commitment. The polypeptides synthesized during phases II and III were first analyzed by one-dimensional polyacrylamide gel electrophoresis. The patterns are similar for the two phenotypes. The major polypeptides synthesized during phase II are also synthesized during phase III, but in addition, a group of at least four new major polypeptides appear during phase III for both phenotypes. The minor polypeptides synthesized during phase III were also compared between the two phenotypes by two-dimensional polyacrylamide gel electrophoresis. The patterns, including roughly 200 distinguishable polypeptides, were similar. The similarities in the patterns of protein synthesis and the delay in the onset of phase III in mycelium forming cells are discussed in terms of phenotypic commitment. From these considerations, alternate hypotheses for the regulation of fungal dimorphism, in particular, and cell divergence, in general, are proposed.     

Effects of agitation and aeration on the production of extracellular glucose oxidase from a recombinant Saccharomycescerevisiae

A recombinant strain of Saccharomyces cerevisiae, GAL-GO2, was developed to facilitate the production of extracellular glucose oxidase (GOD). The recombinant strain secreted 85% (8.7?U/ml) of the total GOD (10.3?U/ml) produced in shake flask culture. For further enhancement of GOD production, optimization of the speed of agitation and the rate of aeration in a stirred tank fermentor was carried out. Response surface methodology with appropriate statistical experimental design was employed for this purpose. The maximal level of extracellular GOD was achieved when the speed of agitation and the rate of aeration were 420?rpm and 0.25?vvm, respectively. The enzyme production was increased by 74% compared to the level obtained under unoptimized conditions.     

Leukotriene B4 synthesis and metabolism by neutrophils and granule-free cytoplasts.

Copper-deprived sycamore (Acer pseudoplatanus) cells do not excrete molecules of active laccase in their culture medium. In the range of 2-100 micrograms of copper initially present per litre of nutrient solution, the total laccase activity measured in the cell suspensions at the end of the exponential phase of growth was closely proportional to the amount of added copper. However, copper-deprived cells excreted the laccase apoprotein (laccase without copper) at the same rate as copper-supplied cells excreted the active, copper-containing, laccase. When the culture medium was initially supplied with limiting amounts of copper, the active laccase was excreted until all copper molecules were metabolized. Thereafter, the laccase apoprotein was excreted. Consequently, at the end of the exponential phase of growth, the cell supernatants contained a mixture of apoprotein and copper-containing laccase. After purification and concentration, this mixture of copper-containing laccase (blue) and laccase apoprotein (slightly yellow) showed a yellow-green colour. Under copper-limiting culture conditions an equivalent decrease of Type 1, Type 2 and Type 3 Cu2+ was observed. Addition of copper to copper-deficient enzyme solutions does not result in a recovery of the enzyme activity. However, when added to copper-deficient sycamore-cell suspensions, copper induced a recovery of the excretion of active enzyme, at a normal rate, within about 10 h. The first molecules of active laccase were excreted after 3-4 h.     

Specific and nonspecific adhesion in the process of retinal cellular aggregation in chick embryos

A study of aggregation of the retinal cells of 8 and 14 day old chick embryos has revealed two phases in this process. The first phase includes the decrease in the concentration of single cells and the increase in the concentration of aggregates. During the second phase the concentration of aggregates falls at the expense of fusion of smaller aggregates into larger ones. The rate of aggregation at both these phases increases with the initial density of cells and decreases with the age of donor embryos and at a suboptimal temperature of cultivation. Aggregation during the first phase does not depend on the presence in the culture medium of divalent cations and colchicine, the level of protein and RNA synthesis in the cells, whereas aggregation during the second phase depends on all these factors. Comparison of these results with the published data suggests that the retinal cell aggregation during the second phase, unlike the first one, is based on the specific adhesiveness of the cells, which is realized via adhesion molecules resynthesized at the cell surface.     

Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris

We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489-1882bp fragment, close to the size of glucose oxidase (GOD), which is 1818bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS-PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing beta-d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 degrees C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.     

Simian virus 40 induces multiple S phases with the majority of viral DNA replication in the G2 and second S phase in CV-1 cells

The infection of permissive monkey kidney cells (CV-1) with simian virus 40 induces G1 growth-arrested cells into the cell cycle. After completion of the first S phase and movement into G2, mitosis was blocked and the cells entered another DNA synthesis cycle (second S phase). Growth-arrested CV-1 cells replicated significant amounts of viral DNA in the G2 phase with the majority of synthesis occurring during the second S phase. When mimosine-blocked (G1/S) infected cells were released into the cell cycle, a major portion of the viral DNA was detected in G2 with the largest accumulation in the second S phase. The total DNA produced per infected cell was 10-12C with approximately 0.5-2C of viral DNA replicated per cell. Therefore the majority of the DNA per cell was cellular, 4C from the first S phase and approximately 4-6C from the second cellular synthesis phase.     

Purification of β-Glucosidase from Saccharomyces lactis Strain Y-123

Saccharomyces lactis strain Y-123, a constitutive high producer of beta-glucosidase (B(h)), was grown in an enriched medium. beta-Glucosidase, extracted most easily by cell autolysis, was purified by successive ammonium sulfate precipitation, ethyl alcohol precipitation, gel filtration, calcium phosphate gel adsorption-elution, and hydroxyapatite column chromatography. The specific activity of the enzyme increased 200-fold during the purification. The electrophoretic and catalytic properties of the enzyme did not change during the procedure. Polyacrylamide gel disc electrophoresis of the partially purified enzyme revealed one major and several minor protein-staining bands. beta-Glucosidase activity in the polyacrylamide gel columns was measured directly by assaying sections of columns frozen and sliced immediately after electrophoresis. Most of the activity coincided with the major protein-staining band. Prolonged assay produced minor activity coinciding with the less intense protein bands. Properties of the enzyme suggest that it is a single, unconjugated, intracellular, high molecular weight protein. The purification procedure is applicable to strains of S. lactis which possess alleles of the B locus for beta-glucosidase synthesis.     

Sulfation and constitutive secretion of dopamine beta-hydroxylase from rat pheochromocytoma (PC12) cells

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.     

Isolation and Purification of Ascorbate Oxidase from Acremonium sp. HI-25

A screening test was undertaken to isolate microorganisms that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of a fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions, optimum conditions for enzyme production were found; strain HI-25 was aerobically cultured by a jar fermenter at 25°C in a medium containing 5% glycerol, 2% defatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH₂PO₄, 0.02% MgSO₄ ·7H₂O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80,000 by SDS polyacrylamide gel electrophoresis and 76,000 by native gel filtration. This enzyme was most active at pH 4.0, 45°C, and was most stable between pH 6.0–10.0 and at temperatures below 60°C.     

Production of thermophilic α-amylase using immobilized transformed Escherichia coli by addition of glycine

Efficient production of thermophilic α-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with κ-carrageenan by the addition of glycine. The effects of glycine, the concentrations of κ-carrageenan and KCI on the production of the enzyme as well as the stability of plasmid pHI301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of α-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term “release”, while diffusion-out from gel beads is referred to by the term “liberate”.) Concentrations of KCI and immobilizing support significantly affected on the liberation of α-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCI, which is a hardening agent for carrageenan. Although the viability of cells and α-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of α-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.     

beta-Glucosidase of Penicillium funiculosum. I. Purification

A strain of Penicillium funiculosum, isolated in this laboratory, produced in high yield both endo- and exo-glucanases and beta-glucosidases, which were suitable for the saccharification of cellulosic materials. The isolation of the beta-glucosidase of this organism, which differs from other beta-glucosidases of fungi in its substrate specificity, by preparative electrophoresis, is described in this article. The organism was grown on a lactose-casein medium and the culture filtrate concentrated by ammonium sulfate precipitation and dialysis. Electrophoresis was carried out on large slabs of polyacrylamide gel in an anodicrun in the presence of borate at pH 7. After elution of active fractions, a cathodic run was made at pH 6.0. Two precipitations with ammonium sulfate resulted in a homogeneous enzyme (specific activity 174 IU/mg). A second isozyme was also produced by P. funiculosum on cellulose-wheat bran medium. This isozyme was purified by electrophoresis at pH 7.0 in the absence of borate and was obtained free from other isozymes of beta-glucosidase and cellulases.