Utilization of oilseed residues and oat bran as substrates for Beta-glucosidase production by Zygomycetous fungi

Utilization of agro- and food-industrial residues has special importance today. One possible way of the applications is the production of industrially interesting microbial hydrolases, for which, solid-state fermentation is a low-cost and environmental friendly biotechnological technique. Since most filamentous fungi are able to biodegrade the cellulosic content of these natural materials, high-yield production of extracellular P-glucosidases can be obtained during the fermentation. In this study, zygomycetous fungal strains representing Rhizomucor miehei, Rhizopus stolonifer, Mucor corticolus, Mortierella echinosphaera and Umbelopsis autotrophica were grown in solid cultures containing various oilseed residues and oat bran as carbon sources to assess the production of their P-glucosidases. Among the oilseed residues, pumpkin seed proved to be the best inductor for P-glucosidase activity. Enzyme production of R. miehei and U. autotrophica was further enhanced when oat bran was used as support. Effect of minerals salts on the enzyme yield was also assayed, in which P-glucosidase production of the investigated strains was generally stimulated after moisturizing the substrates by mineral salt solution

A karotinoid termelés genetikai hátterének vizsgálata genetikailag módosított járomspórás gombák segítségével. = Molecular study of the cartenoid biosynthetic pathway using genetically modified zygomycetous fungal strains

A pályázati program célja, hogy rekombináns technikákat, valamint a genetikai transzformáció különféle eljárásait alkalmazva járomspórás gombák karotinoid bioszintézisét módosítsuk, továbbá, hogy az így létrehozott rendszerben vizsgáljuk a bioszintetikus út lefolyását és a produkció mértékét. Célunk volt, hogy megvizsgáljuk a b-karotinból kiinduló asztaxantin szintézis lehetőségét Mucor circinelloidesben, valamint karotintúltermelő mutánsok előállítása. Mintegy húsz módosult karotinösszetételű (asztaxantin és/vagy kantaxantin termelésre képes), illetve karotin túltermelő M. circinelloides törzset állítottunk elő és jellemeztünk részletesen. Azonosítottuk és részletesen jellemeztük a Rhizomucor miehei HMG-KoA reduktáz, valamint a M. circinelloides izopentenil pirofoszfát izomeráz génjét. Nagyszámú, a karotintermelés módosítását lehetővé tevő transzformációs vektort készítettünk, melyek a további törzsnemesítésben felhasználhatók. 20 járomspórás gombafaj különböző, vad típusú törzseinek karotintermelését elemeztük, különböző környezeti feltételek mellett. Több nagy karotinoid produkcióra képes, illetve új karotinösszetételű törzset azonosítottunk, melyek a további munkákban felhasználhatók. Új, a későbbi alap- és alkalmazott kutatásokban is alkalmazható eljárásokat, protokolokat dolgoztunk ki elsősorban a járomspórás gombák genetikai tramszformációjára, a karotin termeltetés körülményeire (tenyésztés, fermentáció) és a termelés fokozására vonatkozóan. | The objectives of the project were the followings: development of carotene superproducing strains by means of the over-expression of the mevalonate-isoprenoid pathway; introduction of heterologous genes into Mucor circinelloides in order to achieve the production of oxygenated b-carotene derivatives; and extension of these studies to other carotene producer Zygomycetes. Isopentenyl pyrophosphate isomerase gene (McIPP) of Mucor encoding a key-enzyme of the isoprenoid pathway was cloned and analysed. For the modification of the mevalonate pathway, functional analysis of the HMG-CoA reductase gene (hmg) of Rhizomucor miehei was completed and transforming vectors were constructed. Transformation experiments with plasmids harbouring the genes: hmg; isoA (FPP synthase); carG (GGPP synthase) and McIPP, were carried out and several strains with elevated carotene production were obtained. The crtW and crtZ genes from Agrobacterium aurantiacum were placed under the control of M. circinelloides expression signals and heterologous transformations were performed on a selected M. circinelloides strain. Transformants able to produce astaxanthin, cantaxanthin and/or zeaxanthin and β-cryptoxanthin were isolated. Agrobacterium tumefaciens-mediated transformation systems allowing integrative transformation were elaborated and optimized for M. circinelloides and other related carotenoid producer species (other Mucor, Gilbertella and Backusella species)

Production of industrial enzymes in solid state fermentation of agricultural wastes by zygomycetes

Large amount of corn-stalks and leaves arise as agricultural waste during the agro-industrial processes. Solid-state fermentation to produce different industrial enzymes such as cellulolytic enzymes, lipases and proteases, and bioconversion to ethanol may provide an alternative and economic path to utilize these residues. Zygomycetes fungi have been assumed to play an important role in the decomposition of plant and other organic materials in consequence of their effective extracellular enzyme production. Several members of them are commonly used in different biotechnological applications for the large scale production of industrial enzymes, however, production of these enzymes on agricultural wastes are poorly characterized until to date. The aim of our present study was to investigate the activities of cellulolytic enzymes and the production of lipase and protease by zygomycetous strains on agro-industrial wastes such as corn-stalks, corn-leaves and wheat bran. The Mucor, Rhizomucor, Gilbertella and Rhizopus isolates selected for this study have proven to be good extracellular β-glucosidase producer in our previous experiments in which submerged cultivation on cellobiose and solid-state fermentation on wheat bran were used (TAKÓ et al, 2010). In these studies, solid-state fermentation generally resulted in significantly higher enzyme activities than the liquid cultures. Therefore, two solid state fermentation systems were used in the present assays: mixture of chopped corn stalks and corn leaves, and their mixtures with wheat bran at a ratio of 1:1. Fermentations were carried out for 12 days at 25 °C or 37 °C, and enzyme activities were determined from the crude water extracts obtained every second day of the cultivation. Isolates grew intensively on these substrates, and high activities of the cellulolytic enzymes and lipase have been observed during the fermentation period. The total cellulolytic activity of the crude extracts was determined by using Whatman No. 1 filter paper as substrate; in case of each isolate, the highest amount of reducing sugar was measured at the second day of the fermentation. It is worth to point out that significantly higher total cellulolytic activity was usually detected in the crude extracts derived from fermentation medium containing wheat bran. It might be due to the fact that wheat bran supplied convenient amount of nutrients and porosity for oxygen supply. In both fermentation systems, cellobiohydrolase activity of the tested fungi was found to be significantly lower than their endoglucanase and β-glucosidase activities; additionally, it was detected that the activity of endoglucanase generally reaches its maximum during the first half of the incubation, while β-glucosidase on the sixth day or later. Among the tested representatives of the abovementioned genera, a Rhizomucor sp. and a Rhizopus sp. strains proved to be outstanding in its lipase, and protease producing ability, respectively. The investigated fungi could potentially be applicable for biodegradation of agro-industrial wastes and efficient production of industrial enzymes on these cheap, easily available substrates

Utilization of oilseed residues and oat bran as substrates for Beta-glucosidase production by Zygomycetous fungi

Utilization of agro- and food-industrial residues has special importance today. One possible way of the applications is the production of industrially interesting microbial hydrolases, for which, solid-state fermentation is a low-cost and environmental friendly biotechnological technique. Since most filamentous fungi are able to biodegrade the cellulosic content of these natural materials, high-yield production of extracellular P-glucosidases can be obtained during the fermentation. In this study, zygomycetous fungal strains representing Rhizomucor miehei, Rhizopus stolonifer, Mucor corticolus, Mortierella echinosphaera and Umbelopsis autotrophica were grown in solid cultures containing various oilseed residues and oat bran as carbon sources to assess the production of their P-glucosidases. Among the oilseed residues, pumpkin seed proved to be the best inductor for P-glucosidase activity. Enzyme production of R. miehei and U. autotrophica was further enhanced when oat bran was used as support. Effect of minerals salts on the enzyme yield was also assayed, in which P-glucosidase production of the investigated strains was generally stimulated after moisturizing the substrates by mineral salt solution

Screening for Extracellular Lipase Enzymes with Transesterification Capacity in Mucoromycotina Strains

U ovom je radu ispitana hidroliza tributirina pomoću izvanstanične lipaze izolirane iz 169 sojeva plijesni iz razreda Zygomycetes, uključujući i izolate otporne na niske temperature. Od izoliranih se sojeva njih 19 isticalo u proizvodnji enzima, jer su formirali najveće zone lipolitičke aktivnosti oko kolonija. Za daljnje ispitivanje proizvodnje lipolitičkih enzima odabrani su sljedeći sojevi: Mortierella alpina, M. echinosphaera, Mucor corticolus, Rhizomucor miehei, Rhizopus oryzae, Rh. stolonifer, Umbelopsis autotrophica, U. isabellina, U. ramanniana var. angulispora and U. versiformis. Ispitan je utjecaj dodatka Tweena 80, palminog, sojinog, suncokretovog, maslinovog, ekstradjevičanskog maslinovog, sezamovog i bučinog ulja, te ulja pšeničnih klica, kukuruznih klica i sjemenki pamuka na aktivnost enzima. Osim toga, ispitana je i proizvodnja enzima pri submerznom uzgoju te fermentaciji na čvrstoj podlozi od pšeničnih mekinja. Tween 80 i maslinovo ulje učinkovito su potakli proizvodnju lipolitičkih enzima, a pozitivan je učinak imala i podloga od pšeničnih mekinja. Dodatkom mineralnih soli i maslinovog ulja u čvrstu podlogu poboljšana je aktivnost enzima sirovog ekstrakta najmanje 1,5 puta. Ispitana je aktivnost odabranih lipaza u organskoj sintezi, a najbolja je aktivnost postignuta tijekom transesterifikacije p-nitrofenil-palmitata s etanolom pomoću enzima izoliranih iz plijesni R. miehei, Rh. stolonifer i M. echinosphaera.In this study, 169 zygomycetes fungal strains including some cold-tolerant isolates were screened for their extracellular lipolytic activity towards tributyrin. Nineteen of them were outstanding in their enzyme production as they developed the largest lipolytic halo around the colonies in plate tests. Mortierella alpina, M. echinosphaera, Mucor corticolus, Rhizomucor miehei, Rhizopus oryzae, Rh. stolonifer, Umbelopsis autotrophica, U. isabellina, U. ramanniana var. angulispora and U. versiformis were selected for further studies to characterise their lipolytic enzyme production in detail. In these assays, effect of Tween 80 and palm, soybean, sunflower, olive, extra virgin olive, wheat germ, corn germ, sesame seed, pumpkin seed and cottonseed oils on the enzyme activities was investigated, and wheat bran-based submerged and solid-state fermentations were also tested. Tween 80 and olive oil proved to be efficient inductors for lipolytic enzyme production, which was also enhanced when wheat bran was used as support. Addition of mineral salts and olive oil to the solid fermentation medium resulted in at least 1.5-fold increment in the enzyme activities of the crude extracts. Organic synthesis was also assayed by the selected lipases, in which enzymes from the fungi R. miehei, Rh. stolonifer and M. echinosphaera gave the best yields during transesterification reactions between p-nitrophenyl palmitate and ethanol