Studies on methanol — oxidizing yeast

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.     

Bioconversion of methanol to formaldehyde. II. By purified methanol oxidase from modified yeast, Hansenula polymorpha

Modified methylotrophic yeast Hansenula polymorpha (HP A16) that was obtained by repressing leucine oxotrophic yeast; a wild type of Hansenula polymorpha CB4732 was used in this study. The yeast is grown with methanol, which is used as a sole carbon source, using various methanol concentrations and temperatures, and methanol oxidase (MOX) which is a key enzyme of methanol metabolism; production is maximized. Whole yeast cells were cultivated under optimized inoculation conditions; they were separated into two portions. One portion of these cells was directly used in bioconversion of methanol to formaldehyde. The second portion of the free cells was broken into pieces and a crude enzyme extract was obtained. The MOX enzyme in this extract was purified via salt precipitation, dialysis, and chromatographic methods. The purified MOX enzyme of yeast (HP A16) oxidized the methanol to formaldehyde. Optimization of bioconversion conditions was studied to reach maximum activity of enzyme. The optimum temperature and pH were found to be 35 degrees C and pH 8.0 in boric acid/NaOH buffer, and it was stable over the pH range of 6-9, at the 20 degrees C 15 min. A suitable reaction period was found as 50 min. The enzyme indicated low carbon primary alcohols (C2 to C4), as well as methanol. Initially, MOX activity increased with the increase of methanol concentration, but enzyme activity decreased. The apparent Km and Vmax values for methanol substrate of HP A16 MOX were 0.25 mM and 30 U/mg, respectively. The purified MOX enzyme was applied onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis; molecular weight of the enzyme was calculated to be about 670 kDa. Each MOX enzyme is composed of eight identical subunits, each of whose molecular weight is around 82 kDa and which contain eight moles of FAD as the prosthetic group, and the pI of the natural enzyme is found to be 6.4. The purified MOX enzyme was used in the bioconversion of methanol to formaldehyde as a catalyst; this conversion was compared to the conversion percentages of whole cells in our previous article in terms of catalytic performances.     

Increasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut(+)) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15 degrees C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.     

Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeastHansenula polymorpha

Summary Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeastHansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures ofH. polymorpha, equivalent to 35–40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract. it became clear that the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8–9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinantH. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20–22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.Part of these results have been presented at the Deuxième Congrès de la Société Française de Microbiologie, Strasbourg, France, September 1989.     

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G₄S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut⁺) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.     

Toxicity of mercaptoethanol to mutant strains of the yeast Pichia methanolica growing on different carbon sources

Addition of beta-mercaptoethanol at a concentration of 2-3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeast Pichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1. The mutation mth1 of P. methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity. beta-Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions. The growth effect of beta-mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.     

酵母菌辅酶Q提取方法的改进及在假丝酵母属分类学研究中的应用*

本文根据普通实验室的条件,对酵母菌辅酶Q（CoQ）的提取方法进行了改进,既有效地节约了试剂,又避免了一些非常规仪器的使用,使得CoQ分子类型的测定简便易行。用改进的方法,测定了37株假丝酵母属（CandidaBerkhout）菌株的CoQ类型,并据此解决了一些根据常规的形态和生理生化性状难以作出精确鉴定的疑难菌株的分类学问题。     

酵母菌辅酶Q提取方法的改进及在假丝酵母属分类学研究中的应用

本文根据普通实验室的条件,对酵母菌辅酶Q(CoQ)的提取方法进行了改进,既有效地节约了试剂,又避免了一些非常规仪器的使用,使得CoQ分子类型的测定简便易行.用改进的方法,测定了37株假丝酵母属(Candida     

Cytochemical study of catalase activity in Candida boidinii during incubation on methanol

Catalase activity of the methanol-assimilating yeast Candida boidinii M-363 was determined cytochemically and biochemically. Electron microscopic investigations on ultrathin sections were made on cells from 16, 24, and 48h batch cultures in nutrient medium with methanol (or glucose as a control) as the sole source of carbon and energy. The electron-dense oxidation product of 3,3′-diaminobenzidine was found predominantly in the mitochondrial cristae and membranes. The mitochondria were increased in number, enlarged, sometimes aggregated, with variable form and size and they characteristically developed when the strain was grown on methanol. The significant development of these organelles and their intensive DAB staining correlated with the considerable increase in catalase activity. Biochemically, catalase in the cell-free extract was determined to be maximal along the exponential growth phase of the strain during its incubation on methanol. Enzyme analysis of the heavy mitochondrial fraction showed that it possessed catalase activity but not peroxidase activity. The results showed that not only peroxisomes but also mitochondria may be structurally and functionally responsible for the high catalase activity of some methanol-assimilating yeasts. What is more, the contribution of the mitochondria to the utilization of methanol may be significant.     

Chromium in biological systems, I. Some observations on glucose tolerance factor in yeast

Glucose tolerance factor (GTF) has been isolated from a commercially available yeast extract powder, by a simple procedure under mild conditions. This cationic yellow material enhances considerably CO2 production in several yeast strains, after a lag time which can be eliminated by preincubation with glucose. The enhancement of CO2 production by GTF is not specific for glucose, and its effect on galactose raises the possibility that it influences the transport of the sugar to the cells. The ineffectiveness of GTF on cell free extract and the results of a Michaelis plot for CO2 production support this hypothesis.     

Use of malt extract as a nitrogen source for continuous cultivation of Candida utilis]

The effect of an aqueous extract from malt sprouts on the growth and development of fodder yeast was investigated during their continuous cultivation. The extract can be used as a nitrogen source during continuous cultivation of fodder yeast. It is supposed that the extract contains compounds capable to inhibit yeast growth. The extract should be added to the nutrient medium in the amount of 4 g/l of dry weight.     

Isolation from yeast of a metabolically active water-soluble form of ergosterol

A water-soluble complex containing ergosterol together with a component of yeast has been isolated. The complex can be isolated from commercial yeast extract to which ergosterol has been added or directly from whole yeast cells. The complexing component has the properties of a large polysaccharide and the binding between the sterol and the polysaccharide appears to be noncovalent. The complex is easily prepared and is stable in aqueous solution; ergosterol in this solution is metabolically available to yeast cells to which it is added.     

Pharmaco-toxicological study of Kageneckia oblonga, Rosaceae

The probable antipyretic, antiinflammatory, analgesic and antioxidant properties of Kageneckia oblonga, Rosaceae, were investigated and the major compounds of its active extracts were isolated. The study comprised the acute toxicity of the extracts of global methanol, hexane, dichloromethane and methanol. The cytotoxicity of global methanol extract was studied in three tumoral cell lines. All the extracts exhibited the pharmacological activities under study. Methanol and dichloromethane were the most toxic extracts. From the global methanol extract, isolations were performed of prunasin, 23,24- dihydro-cucurbitacin F, and a new cucurbitacin, 3beta-(beta-D-glucosyloxy)-16alpha,23alpha-epoxycucurbita-5,24-diene-11-one. The cytotoxicity of both cucurbitacins on human neutrophils at the assayed concentrations was not statistically significant. In-vitro assays showed that both cucurbitacins can be partly responsible for the analgesic, antipyretic, and anti-inflammatory activities. Evaluation was done of the cytotoxicity of global methanol extract, 23, 24-dihydrocucurbitacin F, aqueous extracts and prunasin against P-388 murine leukaemia, A-549 human lung carcinoma and HT-29 colon carcinoma. Since global methanol extract presented a strong cytotoxicity against P-388 murine leukaemia, A-549 human lung carcinoma, and HT-29 cell lines, it is highly probable that this extract contain one or more cytotoxic compounds that could be investigated for their potential use as an agent against cancer.     

The induction of mycelial development inAureobasidium pullulans (IMI 45533) by yeast extract

Germ tube and subsequent mycelial development from yeast-like and swollen cells ofAureobasidium pullulans (IMI 45533) was induced by yeast extract in defined liquid medium. This morphogenetic transition was dependent on inoculum size; pH effects were not involved and once mycelial development was induced in the cells it continued even in the absence of yeast extract. The progeny of mycelium and future generations were unaffected by yeast extract. Cessation of germination was not due to any obvious medium changes but appeared to be partly due to the production of a germination inhibitor, which could also be produced by control cells grown in the absence of yeast extract.     

比生长速率和氮源对毕赤酵母生产重组鲈鱼生长激素的影响

对毕赤酵母胞内表达重组鲈鱼生长激素(rljGH)的发酵罐上生产进行了研究.建立了指数流加甲醇的策略并考察了不同比生长速率对rljGH生产的影响.结果表明,随着比生长速率的增加,平均比生产速率相应增加,但是胞内持续积累rljGH的时间减少.最大比rljGH产量(0.58 mg/g WCW)在比生长速率为0.029/h时获得.进一步考察了在诱导阶段添加硫酸铵、蛋白胨和酵母抽提物的影响.结果表明,添加硫酸铵和蛋白胨对于rljGH生产没有显著影响;添加2.5 g/L酵母抽提物有助于胞内rljGH的积累,并使胞内积累持续时间由17 h增加到23 h,提高了发酵稳定性.     

Photoreactivation of Transforming DNA by an Enzyme from Bakers' Yeast

Ultraviolet-inactivated Hemophilus influenzae transforming DNA recovers its activity when mixed with cell-free extracts of bakers' yeast and exposed to visible light. The active agent in the extract is not used up in the reaction, and purification has not separated it into more than one non-dialyzable component. It differs from the agent in Escherichia coli extract, which produces very similar photoreactivation, but which can be resolved into non-dialyzable and dialyzable components, the latter being used up during illumination. The yeast agent can be salted out of solution and recovered quantitatively; it is inactivated by crystalline trypsin and chymotrypsin and by brief heating at 60°C.—all facts suggesting that it is an enzyme for which ultraviolet lesions in the DNA serve as substrate. The kinetics of recovery are also consistent with such an assumption. This enzyme is unusual both because it is involved in a light-dependent reaction and because it has a non-destructive action on DNA outside an intact cell.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Cytochemical study of catalase activity in Candida boidinii during incubation on methanol

Catalase activity of the methanol-assimilating yeast Candida boidinii M-363 was determined cytochemically and biochemically. Electron microscopic investigations on ultrathin sections were made on cells from 16, 24, and 48h batch cultures in nutrient medium with methanol (or glucose as a control) as the sole source of carbon and energy. The electron-dense oxidation product of 3,3-diaminobenzidine was found predominantly in the mitochondrial cristae and membranes. The mitochondria were increased in number, enlarged, sometimes aggregated, with variable form and size and they characteristically developed when the strain was grown on methanol. The significant development of these organelles and their intensive DAB staining correlated with the considerable increase in catalase activity. Biochemically, catalase in the cell-free extract was determined to be maximal along the exponential growth phase of the strain during its incubation on methanol. Enzyme analysis of the heavy mitochondrial fraction showed that it possessed catalase activity but not peroxidase activity. The results showed that not only peroxisomes but also mitochondria may be structurally and functionally responsible for the high catalase activity of some methanol-assimilating yeasts. What is more, the contribution of the mitochondria to the utilization of methanol may be significant.     

Determination of tripdiolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reversed-phase high-performance liquid chromatography

Extracts of Tripterygium wilfordii Hook F. have been widely used in China to treat a variety of autoimmune and inflammatory diseases. The diterpenoids triptolide and tripdiolide are two major active components in the T. wilfordii ethyl acetate extract. An efficient solid-phase extraction and high-performance liquid chromatography (SPE-HPLC) method to measure triptolide content in the extract has been previously reported. However, a suitable means of tripdiolide quantification is not available because of interfering compounds in the extract that co-elute with tripdiolide. Therefore, this paper describes a method wherein tripdiolide content can be measured from a small amount of the extract. The extract solution (600 microL) was applied into an aminopropyl SPE tube. Triptolide was eluted with dichloromethane:methanol (1 mL, 49:1 v/v), followed by tripdiolide elution with dichloromethane:methanol (3 mL, 17:3 v/v). The tripdiolide eluate was analysed by HPLC using an isocratic solvent system and was quantified by measuring the peak area at 219 nm. The contents of triptolide and tripdiolide in the extract were determined to be 807.32 +/- 51.94 and 366.13 +/- 17.21 microg/g of extract, respectively. Since tripdiolide is biologically active and makes up a considerable portion of the extract, for extract quality control and standardisation purposes, it should be measured along with triptolide using the proposed SPE-HPLC method.