Utilization of 11β-hydroxysteroids by the salivary gland ducts

Summary Parotid, submandibular and sublingual glands were removed from rats and investigated histochemically. Pyruvate oxidase, iso-citric dehydrogenase, -ketoglutarate oxidase, succinic dehydrogenase, malate dehydrogenase and furfuryl alcohol dehydrogenase activity were observed in the salivary ducts which may be interpreted as significant of high metabolic activity.The 11 -hydroxysteroid dehydrogenase in these ducts displayed marked substrate specificity utilizing 11-hydroxyandrostenedione and cortisol but not 11 -hydroxyoestrone or 11 -hydroxyprogesterone. The relationship between corticosteroids and salivary electrolyte concentrations is discussed.     

Production of interferon-α in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins

Escherichia coli TG1 transformed with a temperature-regulated interferon-α expression vector was grown to high cell density in defined medium containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to achieve an exponential increase in biomass at growth rates which minimized acetate production. Thermal induction of such high cell density cultures resulted in the production of ∼4 g interferon-α/l culture broth. Interferon-α was produced exclusively in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose. The yield of purified interferon-α was ∼300 mg/l with respect to the original high cell density culture broth (overall yield of ∼7.5% active interferon-α). The purified recombinant interferon-α was found by different criteria to be predominantly monomeric and possessed a specific bioactivity of ∼2.5 × 10⁸ IU/mg based on viral cytopathic assay. Received: 8 October 1999 / Received revision: 8 December 1999 / Accepted: 12 December 1999     

Production of α-linolenic acid in Yarrowia lipolytica using low-temperature fermentation

α-Linolenic acid (ALA) is an essential ω-3 fatty with reported health benefits. However, this molecule is naturally found in plants such as flaxseed and canola which currently limits production. Here, we demonstrate the potential to sustainably produce ALA using the oleaginous yeast Yarrowia lipolytica. Through the use of a recently identified Δ12–15 desaturase (Rk Δ12–15), we were able to enable production in Y. lipolytica. When combined with a previously engineered lipid-overproducing strain with high precursor availability, further improvements of ALA production were achieved. Finally, the cultivation of this strain at lower temperatures significantly increased ALA content, with cells fermented at 20 °C accumulating nearly 30% ALA of the total lipids in this cell. This low-temperature fermentation represents improved ALA titer up to 3.2-fold compared to standard growth conditions. Scale-up into a fed-batch bioreactor produced ALA at 1.4 g/L, representing the highest published titer of this ω-3 fatty acid in a yeast host.

     

Capture of bacteria from fermentation broth by body feed filtration: a solved problem?

The direct capture of bacteria produced in high cell density fermentation by filtration is not possible once the milliliter-scale has been surpassed. Filtration in the presence of a filter aid (body feed filtration) constitutes a putative and scalable alternative, but only if conditions proposed by industry for large-scale filtration processes, namely, flow rates (for aqueous solutions) in the range of 500-1,500 L/(m(2) x h) and a filter aid concentration of 相似文献   

Production of β-carotene from deproteinized waste whey filtrate using Mucor azygosporus MTCC 414 in submerged fermentation

The cheese whey, a by-product of dairy industry proved to be an attractive substrate for production of β-carotene. The β-carotene production from Mucor azygosporus MTCC 414 by using deproteinized waste whey filtrate under submerged fermentation was investigated. Various fermentation variables, such as lactose content in whey, initial pH, production temperature, incubation time, and carbon and nitrogen sources played significant role on β-carotene production. Maximum β-carotene production (385 μg/g dcw) was obtained with the whey (pH 5.5) containing 3.5% (w/v) lactose supplemented with soluble starch at (1.0%, w/v) at 30°C after a 5 days incubation. Moreover, unlike other microorganisms which utilize pre-hydrolyzed lactose, this Mucor azygosporus MTCC 414 was found to be capable of utilizing unhydrolyzed lactose present in the whey.     

Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris

Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

11α-Hydroxylation of 16α,17-epoxyprogesterone by Rhizopus nigricans in a biphasic ionic liquid aqueous system

11α-Hydroxylation of 16α,17-epoxyprogesterone (EP) by Rhizopus nigricans is an essential step in the synthesis of many steroidal drugs, while low conversion of the biohydroxylation is a tough problem to be solved urgently in industry. Two ionic liquids (ILs) of [BMIm][PF(6)] and [BMIm][NTf(2)] were used in the biotransformation of EP by R. nigricans. The results indicated that the conversion carried out in [BMIm][PF(6)]-aqueous biphasic system was greatly increased to above 90% at 18 g/L feeding concentration. A simplified mechanism was proposed to explain the improvement of the bioconversion in a biphasic ionic liquid aqueous system. Besides, successive three batches of bioconversion were carried out in the biphasic system with a total conversion of 87% at phase ratio 10 and 75% at phase ratio 5, respectively. Since recycling of the [BMIm][PF(6)] is quite easy, there is a great potential for the application of ILs in fungi biotransformation to implement green production.     

Production of 5′-dRiboflavin-α-d-glucopyranoside in Growing Cultures by the Mold Mucor

During the investigations on riboflavin glycoside formation by Aspergillus, Mucor, Penicillium and Rhizopus, a remarkable production of 5′-d-riboflavin-α-d-glucopyranoside was observed in several strains belonging to the genus Mucor when grown on a, medium containing maltose and riboflavin. Several conditions on 5′-d-riboflavin-α-d-glucopyranoside formation were also investigated with washed mycellium of M. javanicus. Maltosyl compounds such as maltose, dextrin, amylose and soluble starch were the effective glucosyl donor, whereas glucose, fructose, sucrose, lactose and dextran were inactive.     

Production of recombinant single chain antibodies (scFv) in vegetatively reproductive Kalanchoe pinnata by in planta transformation

We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins.     

The Production of Volatile Compounds by Yeasts Isolated from Small Brazilian cachaça distilleries

Summary The production of volatile compounds by 24 strains of Saccharomyces cerevisiae and one strain each of Candida apicola, C. famata, C. guilliermondii, Hanseniaspora occidentalis, Pichia subpelicullosa and Schizosaccharomyces pombe was evaluated with respect to the production of cacha?a. They were isolated from small cacha?a distilleries (27), industrial cacha?a distilleries (2) and one sugarcane alcohol distillery, and tested in synthetic medium for the production of acetaldehyde, ethyl acetate, propanol, isobutanol, isoamyl alcohol, acetic acid and glycerol. The Saccharomyces strains showed a narrow range of variation in the production of such compounds, near 50% of the average of each volatile compound concentration. Principal component analysis showed the separation of the strains into six groups, and acetic acid production was the variable of greatest impact in the differentiation of the strains. The strains of S. pombe formed a distinct group (Group 2), and the strains of C. apicola and H. occidentalis formed a joint group (Group 6) as did Sc13 and Sc4 (Group 4). Group 1 was formed exclusively of S. cerevisiae. The closest non-Saccharomyces strains were C. apicola and H. occidentalis, with a similarity index of about 0.95. The strain P. subpelliculosa showed general characteristics more similar to those of the S. cerevisiae strains than to the non-Saccharomyces strains.     

Production and characteristics of an intracellularα-glucosidase from a color variant strain ofAureobasidium pullulans

Aureobasidium pullulans produced an intracellular-glucosidase. The enzyme was purified 124-fold by solubilization with Triton X-100, Q-Sepharose treatment, hydroxylapatite, octyl-Sepharose column chromatography, and gel filtration on Sephacryl S-200, and had a specific activity of 316.82 U/mg protein. The enzyme displayed an optimum pH for its action at 4.0 and was fully stable at pH 3.0–6.0 at 50°C. The-glucosidase was completely stable up to 60°C and had an optimum activity at 60°C. The partially purified enzyme preparation hydrolyzed maltose, isomaltose, sucrose, and trehalose at relative rates of 100, 60, 47, and 50, respectively, and had little or no activity on polysaccharides. TheK _m value for maltose hydrolysis at pH 4.0 and 50°C was 1.85mm. The enzyme was not adsorbed onto raw corn starch and showed little raw starch degradation. The-glucosidase did not require any metal ion for activity. This represents the first characterization of intracellular-glucosidase fromA. pullulans.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Production and characterization of partially purified extracellular thermostable α-amylase by Bacillus subtilis in submerged fermentation (SmF)

A Bacillus strain was isolated from soil samples from the campus area of Dicle University. Based on 16S ribosomal RNA sequencing, the microorganism was closely related to Bacillus subtilis. Effects of different culture medium, incubation time, carbon and nitrogen sources, and various starches, flours, and chemicals on α-amylase production were examined. Maximum enzyme production (7516 U/mL) was obtained in a basal medium A containing 0.05% Tween 40 in 24 h. Partially purified enzyme showed maximum activity at 60 °C with an optimum pH of 6.0. The effects of 0.2% detergents (sodium dodecyl sulfate [SDS], CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], and commercial detergent Omo Matic) on partially purified enzyme activity over a period of time (15-150 min) were examined and the order of inhibition effect from the most to the least was found as SDS > Omo Matic > CHAPS. Different metal ions inhibited α-amylase activity at low concentrations (1.5 mM). Co2? was a mild inhibitor and Hg2? and Cd2? were potent inhibitors, whereas Ca2? and Mg2? increased the enzyme activity. At 20 mM, Ca2? enhanced enzyme activity, and different Ca2? concentrations (10-300 mM) were studied.     

Phosphate effects in the fermentation of α-amylase by Bacillus amyloliquefaciens

Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding.     

Growth of a photosynthetic bacterium anaerobically in darkness,supported by “oxidant-dependent” sugar fermentation

Rhodopseudomonas capsulata can obtain energy for growth from light (anaerobically) and can also grow heterotrophically in darkness using alternative energy conversion modes, namely, aerobic respiration or an unusual type of anaerobic catabolism of sugars. Dark anaerobic growth with fructose as sole carbon and energy source is dependent on the presence of an accessory oxidant such as trimethylamine-N-oxide, is accompanied by production of lactate and other classical fermentation products, and yields cells with a high content of photosynthetic pigments and polyhydroxybutyrate.Abbreviation Used TMAO trimethylamine-N-oxide - DMSO dimethyl sulfoxide - EMP Embden-Meyerhof-Parnas - ED Entner-Doudoroff - PHB polyhydroxybutyrate     

Isolation and Characterization of a β-Glucosidase from a Clavispora Strain with Potential Applications in Bioethanol Production from Cellulosic Materials

We previously reported on a new yeast strain of Clavispora sp. NRRL Y-50464 that is capable of utilizing cellobiose as sole source of carbon and energy by producing sufficient native β-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultaneous saccharification and fermentation. Eliminating the addition of external β-glucosidase reduces the cost of cellulosic ethanol production. In this study, we present results on the isolation and identification of a β-glucosidase protein from strain Y-50464. Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and blast search of the NCBInr database (National Center for Biotechnology Information nonredundant), the protein from Y-50464 was identified as a β-glucosidase (BGL1) with a molecular weight of 93.3 kDa. The BGL1 protein was purified through multiple chromatographic steps to a 26-fold purity (K _m?=?0.355 mM [pNPG]; K _i?=?15.2 mM [glucose]), which has a specific activity of 18.4 U/mg of protein with an optimal performance temperature at 45 °C and pH of 6.0. This protein appears to be intracellular although other forms of the enzyme may exist. The fast growth rate of Y-50464 and its capability to produce sufficient β-glucosidase activity for ethanol conversion from cellobiose provide a promising means for low-cost cellulosic ethanol production through a consolidated bioprocessing development.     

Production of α-amylase in a low cost medium by Bacillus licheniformis TCRDC-B13

A low cost synthetic medium producing large quantities of α-amylase has been developed. Bacillus licheniformis TCRDC-B13 isolated from soil was used for α-amylase production. The α-amylase enzyme of this strain showed excellent stability at high temperatures and over a wide pH range. The low cost medium produced 5 times more enzyme than the high cost synthetic medium (using yeast extract and peptone) in shake flasks. In a 2.6-l fermentor, the enzyme production further doubled.