Mikrobna proizvodnja limunske i izolimunske kiseline na podlozi od suncokretovog ulja

The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Yarrowia lipolytica N 15 as well the biosynthesis of citric and isocitric acids on sunflower oil were studied. It was indicated that cell growth was associated with the simultaneous utilization of glycerol and free fatty acids produced during oil hydrolysis. The activities of enzymes of glycerol metabolism (glycerol kinase), fatty acid assimilation enzymes of glyoxylate cycle (isocitrate lyase and malate synthase) and citric acid cycle were comparatively assayed in Y. lipolytica grown on sunflower oil, glycerol and oleic acid. Glycerol kinase and enzymes of glyoxylate cycle were active during the whole period of cell cultivation on sunflower oil. Citric acid production and a ratio between citric and isocitric acids depended on both the strain used and the medium composition. It was revealed that wild type strain Y. lipolytica VKM Y-2373 produced almost equal amounts of citric and isocitric acids at pH=4.5 and predominantly accumulated isocitric acid at pH=6.0. The mutant Y. lipolytica N 15 produced only citric acid (150 g/L with mass yield (YCA) of 1.32 g/g). Biochemical characteristics of mutant strain Y. lipolytica N 15 were discussed.U radu je ispitan rast divljeg soja kvasca Yarrowia lipolytica VKM Y-2373 i mutantnog soja Yarrowia lipolytica N 15, te biosinteza limunske i izolimunske kiseline na podlozi od suncokretovog ulja. Utvrđeno je da je rast stanica povezan s istodobnim iskorištenjem glicerola i slobodnih masnih kiselina nastalih tijekom hidrolize ulja. Usporedno je ispitana aktivnost enzima koji sudjeluju u metabolizmu glicerola (glicerol kinaze), glioksilatnom ciklusu asimilacije masnih kiselina (izocitrat liaze i malat sintaze) i ciklusu limunske kiseline u kvascu Y. lipolytica uzgojenom na podlozi od suncokretovog ulja, glicerola i oleinske kiseline. Glicerol kinaza i enzimi glioksilatnog ciklusa bili su aktivni cijelo vrijeme uzgoja stanica na podlozi od suncokretovog ulja. Količina proizvedene limunske kiseline i omjer limunske i izolimunske kiseline ovise o upotrijebljenom soju i sastavu podloge. Otkriveno je da je divlji soj Y. lipolytica VKM Y-2373 proizveo skoro jednaku količinu limunske i izolimunske kiseline pri pH=4,5, a pretežno izolimunsku kiselinu pri pH=6.0. Mutantni soj Y. lipolytica N 15 proizveo je samo limunsku kiselinu (150 g/L, s masenim stupnjem pretvorbe YCA=1,32 g/g). U radu su također razmotrene biokemijske značajke mutantnog soja Y. lipolytica N 15

Mikrobna proizvodnja limunske i izolimunske kiseline na podlozi od suncokretovog ulja

The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Yarrowia lipolytica N 15 as well the biosynthesis of citric and isocitric acids on sunflower oil were studied. It was indicated that cell growth was associated with the simultaneous utilization of glycerol and free fatty acids produced during oil hydrolysis. The activities of enzymes of glycerol metabolism (glycerol kinase), fatty acid assimilation enzymes of glyoxylate cycle (isocitrate lyase and malate synthase) and citric acid cycle were comparatively assayed in Y. lipolytica grown on sunflower oil, glycerol and oleic acid. Glycerol kinase and enzymes of glyoxylate cycle were active during the whole period of cell cultivation on sunflower oil. Citric acid production and a ratio between citric and isocitric acids depended on both the strain used and the medium composition. It was revealed that wild type strain Y. lipolytica VKM Y-2373 produced almost equal amounts of citric and isocitric acids at pH=4.5 and predominantly accumulated isocitric acid at pH=6.0. The mutant Y. lipolytica N 15 produced only citric acid (150 g/L with mass yield (YCA) of 1.32 g/g). Biochemical characteristics of mutant strain Y. lipolytica N 15 were discussed.U radu je ispitan rast divljeg soja kvasca Yarrowia lipolytica VKM Y-2373 i mutantnog soja Yarrowia lipolytica N 15, te biosinteza limunske i izolimunske kiseline na podlozi od suncokretovog ulja. Utvrđeno je da je rast stanica povezan s istodobnim iskorištenjem glicerola i slobodnih masnih kiselina nastalih tijekom hidrolize ulja. Usporedno je ispitana aktivnost enzima koji sudjeluju u metabolizmu glicerola (glicerol kinaze), glioksilatnom ciklusu asimilacije masnih kiselina (izocitrat liaze i malat sintaze) i ciklusu limunske kiseline u kvascu Y. lipolytica uzgojenom na podlozi od suncokretovog ulja, glicerola i oleinske kiseline. Glicerol kinaza i enzimi glioksilatnog ciklusa bili su aktivni cijelo vrijeme uzgoja stanica na podlozi od suncokretovog ulja. Količina proizvedene limunske kiseline i omjer limunske i izolimunske kiseline ovise o upotrijebljenom soju i sastavu podloge. Otkriveno je da je divlji soj Y. lipolytica VKM Y-2373 proizveo skoro jednaku količinu limunske i izolimunske kiseline pri pH=4,5, a pretežno izolimunsku kiselinu pri pH=6.0. Mutantni soj Y. lipolytica N 15 proizveo je samo limunsku kiselinu (150 g/L, s masenim stupnjem pretvorbe YCA=1,32 g/g). U radu su također razmotrene biokemijske značajke mutantnog soja Y. lipolytica N 15

Evolution of glyoxylate cycle enzymes in Metazoa: evidence of multiple horizontal transfer events and pseudogene formation

BACKGROUND: The glyoxylate cycle is thought to be present in bacteria, protists, plants, fungi, and nematodes, but not in other Metazoa. However, activity of the glyoxylate cycle enzymes, malate synthase (MS) and isocitrate lyase (ICL), in animal tissues has been reported. In order to clarify the status of the MS and ICL genes in animals and get an insight into their evolution, we undertook a comparative-genomic study. RESULTS: Using sequence similarity searches, we identified MS genes in arthropods, echinoderms, and vertebrates, including platypus and opossum, but not in the numerous sequenced genomes of placental mammals. The regions of the placental mammals' genomes expected to code for malate synthase, as determined by comparison of the gene orders in vertebrate genomes, show clear similarity to the opossum MS sequence but contain stop codons, indicating that the MS gene became a pseudogene in placental mammals. By contrast, the ICL gene is undetectable in animals other than the nematodes that possess a bifunctional, fused ICL-MS gene. Examination of phylogenetic trees of MS and ICL suggests multiple horizontal gene transfer events that probably went in both directions between several bacterial and eukaryotic lineages. The strongest evidence was obtained for the acquisition of the bifunctional ICL-MS gene from an as yet unknown bacterial source with the corresponding operonic organization by the common ancestor of the nematodes. CONCLUSION: The distribution of the MS and ICL genes in animals suggests that either they encode alternative enzymes of the glyoxylate cycle that are not orthologous to the known MS and ICL or the animal MS acquired a new function that remains to be characterized. Regardless of the ultimate solution to this conundrum, the genes for the glyoxylate cycle enzymes present a remarkable variety of evolutionary events including unusual horizontal gene transfer from bacteria to animals. REVIEWERS: Arcady Mushegian (Stowers Institute for Medical Research), Andrey Osterman (Burnham Institute for Medical Research), Chris Ponting (Oxford University)

Biotehnološka primjena kvasca Yarrowia lipolytica uzgojenog u uvjetima nedostatka tiamina

During the cultivation of a thiamine-auxotrophic yeast strain Yarrowia lipolytica VKM Y-2412 on ethanol, the growth limitation by thiamine leads to the production of α-ketoglutaric acid. The α-ketoglutaric acid synthesis has been studied in dependence on pH, oxygen supply and ethanol, zinc and iron concentrations. Under optimal conditions, Y. lipolytica produced 88.7 g/L of α-ketoglutaric acid. The culture broth containing α-ketoglutaric acid was subjected to chemical treatment with hydrogen peroxide, which led to the formation of succinic acid in significant quantities (71.7 g/L). Further direct esterification of succinic acid with excess absolute ethanol yielded diethyl succinate. Biomass of Y. lipolytica, a superproducer of α-ketoglutaric acid, was characterized by a high content of protein and essential amino acids, free amino acids, and unusually large amount of γ-aminobutyric acid. The unique amino acid composition of the producer makes it possible to use this biomass as a component of parenteral nutrition mixtures and as a basis for neuroleptics.Auksotrofni kvasac Yarrowia lipolytica VKM Y-2412 uzgojen je na podlozi s etanolom u uvjetima nedostatka tiamina, što je dovelo do nastajanja α-ketoglutarne kiseline. Ispitana je ovisnost njezine sinteze o pH-vrijednosti, opskrbljenosti kisikom i etanolom, te koncentracijama cinka i željeza. U optimalnim je uvjetima kvasac Y. lipolytica proizveo 88,7 g/L α-ketoglutarne kiseline. Podloga za uzgoj koja je sadržavala α-ketoglutarnu kiselinu kemijski je obrađena vodikovim peroksidom, što je dovelo do nastajanja značajne koncentracije sukcinske kiseline (71,7 g/L). Daljnjom izravnom esterifikacijom sukcinske kiseline uz suvišak apsolutnog etanola dobiven je dietil sukcinat. Biomasu kvasca Y. lipolytica, koji proizvodi velike količine α-ketoglutarne kiseline, karakterizira veliki udjel proteina i esencijalnih aminokiselina, slobodnih aminokiselina i neuobičajeno velika količina γ-aminomaslačne kiseline. Jedinstveni aminokiselinski sastav kvasca omogućuje njegovu primjenu u intravenoznoj prehrani i pri liječenju pacijenata antipsihoticima

Lipase Secretion and Citric Acid Production in Yarrowia lipolytica Yeast Grown on Animal and Vegetable Fat

The aim of the study was to investigate the potentiality of the utilization of raw agro- -industrial fat for the biotechnological production of valuable products (lipase and citric acid) by the yeast Yarrowia (Candida) lipolytica. Thirty strains of the aforementioned species were investigated for their capability of lipase secretion and citric acid production on media containing animal fat or rapeseed oil as a sole carbon and energy source. Strain Y. lipolytica 704, exhibiting the highest lipase activity on rapeseed oil (2760 U/mL), was selected for the study of biochemical peculiarities of cell growth, and strain Y. lipolytica 187/1, exhibiting the maximum citric acid synthesis, was selected for the subsequent studies on citric acid production. A relationship between lipase production and residual rapeseed oil concentration was studied. The essential factor for lipase production was found to be the concentration of rapeseed oil in the medium, which should be no less than 5 g/L. Under optimal conditions of cultivation, citric acid production by rapeseed-oil-grown yeast Yarrowia lipolytica 187/1 amounted to 135 g/L; specific rate of citric acid production reached m(CA)/m(cell)=127 mg/(g·h); mass yield (YCA) and energy yield (hCA) were 1.55 and 0.41, respectively

Proizvodnja limunske kiseline pomoću kvasaca uzgojenih na podlogama s glicerolom, dobivenim pri proizvodnji biodizela

The possibility of using glycerol and glycerol-containing waste from biodiesel manufacture as a carbon and energy source for microbiological production of citric acid has been studied. Acid formation on the selective media had previously been tested in 66 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis and Yarrowia). Under growth limitation by nitrogen, 41 strains (belonging mainly to species Yarrowia lipolytica) produced acids; unlike 25 strains of the genera Debaryomyces, Candida, Pichia, Saccharomyces and Torulopsis. Among the 41 acid-producing strains, mutant strain Yarrowia lipolytica N15 was selected since it was able to produce citric acid presumably in high amounts. The citric acid production by the selected strain was studied in dependence on the medium pH, aeration and concentration of glycerol. Under optimal conditions, the mutant Y. lipolytica N15 produced up to 98 g/L of citric acid when grown in a fermentor with the medium containing pure glycerol, and 71 g/L of citric acid when grown on glycerol-containing waste. The effect of growth phases on physiological peculiarities of the citric acid producer was discussed.Ispitana je mogućnost uporabe glicerola i otpada bogatoga glicerolom, dobivenih pri proizvodnji biodizela, kao izvora ugljika i energije za mikrobnu proizvodnju limunske kiseline. Prethodno je ispitana proizvodnja limunske kiseline na selektivnim podlogama pomoću 66 sojeva kvasaca različitih vrsta (Candida, Pichia, Saccharomyces, Torulopsis i Yarrowia). Pri smanjenim koncentracijama dušika 41 soj (uglavnom vrste Yarrowia lipolytica) mogao je proizvesti limunsku kiselinu, dok 25 sojeva vrsta Debaryomyces, Candida, Pichiа, Saccharomyces i Torulopsis to nije moglo. Od 41 soja odabran je mutant Yarrowia lipolytica N15, jer je mogao proizvesti veliku količinu limunske kiseline. Ispitan je utjecaj pH-vrijednosti podloge, prozračivanja tijekom reakcije i koncentracije glicerola u podlozi na proizvodnju limunske kiseline pomoću toga soja. U optimalnim uvjetima u fermentoru mutant Yarrowia lipolytica N15 proizveo je do 98 g/L limunske kiseline na podlozi sa čistim glicerolom, a do 71 g/L limunske kiseline na podlozi s otpadom bogatim glicerolom. U radu se raspravlja i o utjecaju faze rasta na fiziološke značajke kvasaca pri proizvodnji limunske kiseline

Lipase Secretion and Citric Acid Production in Yarrowia lipolytica Yeast Grown on Animal and Vegetable Fat

The aim of the study was to investigate the potentiality of the utilization of raw agro- -industrial fat for the biotechnological production of valuable products (lipase and citric acid) by the yeast Yarrowia (Candida) lipolytica. Thirty strains of the aforementioned species were investigated for their capability of lipase secretion and citric acid production on media containing animal fat or rapeseed oil as a sole carbon and energy source. Strain Y. lipolytica 704, exhibiting the highest lipase activity on rapeseed oil (2760 U/mL), was selected for the study of biochemical peculiarities of cell growth, and strain Y. lipolytica 187/1, exhibiting the maximum citric acid synthesis, was selected for the subsequent studies on citric acid production. A relationship between lipase production and residual rapeseed oil concentration was studied. The essential factor for lipase production was found to be the concentration of rapeseed oil in the medium, which should be no less than 5 g/L. Under optimal conditions of cultivation, citric acid production by rapeseed-oil-grown yeast Yarrowia lipolytica 187/1 amounted to 135 g/L; specific rate of citric acid production reached m(CA)/m(cell)=127 mg/(g·h); mass yield (YCA) and energy yield (hCA) were 1.55 and 0.41, respectively

Selection of Producer of α-Ketoglutaric Acid from Ethanol-Containing Wastes and Impact of Cultivation Conditions

Ester–aldehyde fraction (EAF) is a by-product of ethyl-alcohol-producing companies whose purification requires an expensive process. The results of this study illustrate the environmentally friendly and alternative possibility of using EAF to increase their value as substrate to produce α-ketoglutaric acid (KGA) using different yeasts. It was found that some species of the genera Babjeviella, Diutina, Moesziomyces, Pichia, Saturnispora, Sugiyamaella, Yarrowia and Zygoascus grown under thiamine deficiency accumulate KGA in the medium with an EAF as the sole carbon source. The strain Y. lipolytica VKM Y-2412 was selected as the producer. To reach the maximum production of KGA, the cultivation medium should contain 0.3 µg/L thiamine during cultivation in flasks and 2 µg/L in the fermentor; the concentration of (NH4)2SO4 should range from 3 to 6 g/L; and the optimal concentrations of Zn2+, Fe2+ and Cu2+ ions should be 1.2, 0.6 and 0.05 mg/L, respectively. EAF concentration should not exceed 1.5 g/L in the growth phase and 3 g/L in the KGA synthesis phase. At higher EAF concentrations, acetic acid was accumulated and inhibited yeast growth and KGA production. Under optimal conditions, the producer accumulated 53.8 g/L KGA with a yield (Yp/s) of 0.68 g/g substrate consumed

Physiological, Biochemical and Energetic Characteristics of Torulaspora globosa, a Potential Producer of Biofuel

It was obtained that the yeast Torulaspora globosa VKPM Y-953 is suitable for the production of biodiesel fuel. Zinc plays an important regulatory role in the metabolism of the studied strain. The study of the growth parameters and the fatty acid profile of the yeast T. globosa showed that the limitation of its growth by ethanol, at different concentrations of zinc in the medium, considerably influences the chemical composition and the energy content of yeast cells, but not their yield by weight (Yx/s). The increased concentrations of zinc in the medium, in combination with the yeast growth limitation by ethanol, elevated the content of lipids in the cells by 28% and diminished the content of proteins by 14%. At the same time, the limitation of yeast growth by zinc decreased Yx/s and energy (ηX/S) by 2.6 and 3.1 times, respectively. In this case, the content of lipids in cells fell by 72%, while that of proteins rose by 65%. The fatty acid profile of the T. globosa cells was used to estimate the main characteristics of biodiesel (iodine value, cetane number, density, and kinematic viscosity). The biomass of T. globosa can also be used in agriculture as a feed additive rich in essential amino acids

Microbiological Production of Citric and Isocitric Acids from Sunflower Oil

The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Yarrowia lipolytica N 15 as well the biosynthesis of citric and isocitric acids on sunflower oil were studied. It was indicated that cell growth was associated with the simultaneous utilization of glycerol and free fatty acids produced during oil hydrolysis. The activities of enzymes of glycerol metabolism (glycerol kinase), fatty acid assimilation enzymes of glyoxylate cycle (isocitrate lyase and malate synthase) and citric acid cycle were comparatively assayed in Y. lipolytica grown on sunflower oil, glycerol and oleic acid. Glycerol kinase and enzymes of glyoxylate cycle were active during the whole period of cell cultivation on sunflower oil. Citric acid production and a ratio between citric and isocitric acids depended on both the strain used and the medium composition. It was revealed that wild type strain Y. lipolytica VKM Y-2373 produced almost equal amounts of citric and isocitric acids at pH=4.5 and predominantly accumulated isocitric acid at pH=6.0. The mutant Y. lipolytica N 15 produced only citric acid (150 g/L with mass yield (YCA) of 1.32 g/g). Biochemical characteristics of mutant strain Y. lipolytica N 15 were discussed