Immobilized Forms of the Ophiostoma piceae Lipase for Green Synthesis of Biodiesel. Comparison with Eversa Transform 2.0 and Cal A

In this work, we analyzed the suitability of a versatile recombinant lipase, secreted by Ophiostoma piceae (OPEr) and produced in Pichia pastoris, as a catalyst of the synthesis of biodiesel. The enzyme was immobilized by five covalent procedures and by hydrophobicity on functionalized nanoparticles of magnetite or of a novel Zn/Mn oxide named G1. Then, they were tested for green production of biodiesel by solventless enzymatic transesterification of discarded cooking oil and methanol (1:4) at 25 °C. The results were compared with those shown by free OPEr and the commercial lipases Eversa® and Cal A®. Several preparations with immobilized OPEr produced high synthesis yields (>90% transesterification), comparable to those obtained with Eversa®, the commercial enzyme designed for this application. Three of the biocatalysts maintained their catalytic efficiency for nine cycles. The process catalyzed by AMNP-CH-OPEr was scaled from 500 µL to 25 mL (50 times), improving its efficiency.

Synergistic Ability of Chitosan and Trichoderma harzianum to Control the Growth and Discolouration of Common Sapstain Fungi of Pinus radiata

An environmentally compatible method for controlling sapstain fungi in wood was evaluated, using a combination of chitosan and an albino strain of Trichoderma harzianum, a biological control agent (BCA). The growth and penetration into the wood of the sapstain fungi Ophiostoma piceae, Leptographium procerum, and Sphaeropsis sapinea were assessed in radiata pine wafers treated with chitosan and BCA, both alone and in combination. Several mycological and microscopic techniques were used, including a gfp (green fluorescent protein) transformed strain of O. piceae for assessing the depth of penetration in the wood samples. The synergy between the chitosan and BCA was evident, and for two tested fungi, only the combination of chitosan and BCA afforded protection. The synnemata (recognized by erect conidiogenous cells bearing conidia) was observed on the surface of the wafers inoculated with L. procerum and O. piceae, but the hyphae were unable to penetrate and melanise. The results suggest that the limited ability of chitosan to penetrate deeply into the wood was compensated by the fast growth of T. harzianum in the inner wood.

Effect of the Immobilization Strategy on the Efficiency and Recyclability of the Versatile Lipase from Ophiostoma piceae

The recombinant lipase from Ophiostoma piceae OPEr has demonstrated to have catalytic properties superior to those of many commercial enzymes. Enzymatic crudes with OPEr were immobilized onto magnetite nanoparticles by hydrophobicity (SiMAG-Octyl) and by two procedures that involve covalent attachment of the protein (mCLEAs and AMNP-GA), giving three nanobiocatalysts with different specific activity in hydrolysis of p-nitrophenyl butyrate (pNPB) and good storage stability at 4 °C over a period of 4 months. Free OPEr and the different nanobiocatalysts were compared for the synthesis of butyl esters of volatile fatty acids C4 to C7 in reactions containing the same lipase activity. The esterification yields and the reaction rates obtained with AMNP-GA-OPEr were in general higher or similar to those observed for the free enzyme, the mCLEAs-OPEr, and the non-covalent preparation SiMAG-Octyl-OPEr. The time course of the esterification of the acids C4 to C6 catalyzed by AMNP-GA-OPEr was comparable. The synthesis of the C7 ester was slower but very efficient, admitting concentrations of heptanoic acid up to 1 M. The best 1-butanol: acid molar ratio was 2:1 for all the acids tested. Depending on the substrate, this covalent preparation of OPEr maintained 80–96% activity over 7 cycles, revealing its excellent properties, easy recovery and recycling, and its potential to catalyze the green synthesis of chemicals of industrial interest.

Assemblages of ophiostomatoid fungi vectored by Ips amitinus (Coleoptera: Scolytinae) on norway spruce depend on colonization time, position on the host tree and development stage

Mali osmerozubi smrekov pisar Ips amitinus najčešće naseljava smreku u montanskima područjima Središnje Europe. Najvažniji domaćin je obična smreka (Picea abies) i bijeli bor (Pinus sylvestris). U ugodnim vremenskim i trofičnim uvjetima, potkornjak postaje opasan, posebno za mlađa stabla u plantažama. Klimatske promjene, s kojima se suočavamo danas, mogu biti povoljne za vrste koje nisu bile ekonomski važne u prošlosti, a u zadnje vrijeme počinju pričinjavati štete u šumama. Informacije o ekološkim/biološkim obilježjima I. amitinus su u literaturi rijetke, posebice za asocijacije potkornjaka i gljiva; iako je poznato da su vrste potkornjaka (Coleoptera: Scolytinae) povezane s različitim gljivama. Istraživali smo čimbenike koji utječu na asocijacije ofiostomatoidnih gljiva s I. amitinus na običnoj smreki. Materijal za studiju bio je prikupljen 2010. godine u blizini Dravograda, na sjeveru Slovenije, gdje je u zimskim vjetrovima bila porušena obična smreka. Za izolacije ofiostomatoidnih gljiva prikupili smo 442 uzorka (kukci i zaraženo drvo - uzorci iz drvenih diskova, s dva stabla na 0,5 m, 6 m i 15 metara iznad panja). Uzeto je ukupno 625 izolata. Ofiostomatoidne gljive su bile najbrojnije zastupljene skupine. Identificirali smo deset vrsta gljiva. Najčešća je bila Ophiostoma brunneo-ciliatum, slijedile su Grosmannia penicillata, Ophiostoma bicolor, Ceratocystiopsis minuta, Grosmannia piceiperda, Endoconidiophora polonica, Ophiostoma piceae, Ophiostoma fuscum, Grosmannia cucullata, Graphium fimbriisporum. Povezanost I. amitinus s O. fuscum, G. cucullata i G. fimbriisporum bila je prvi put potvrđena. Istraživali smo razlike u pojavljivanju pojedinih vrsta gljiva u različitim stadijima života potkornjaka (adulti, ličinke, kukuljice) i zaraženih uzoraka drva.

Quorum-Sensing Mechanisms Mediated by Farnesol in Ophiostoma piceae: Effect on Secretion of Sterol Esterase

ABSTRACTOphiostoma piceaeCECT 20416 is a dimorphic wood-staining fungus able to produce an extracellular sterol-esterase/lipase (OPE) that is of great biotechnological interest. In this work, we have studied the morphological change of this fungus from yeast to hyphae, which is associated with the cell density-related mechanism known as quorum sensing (QS), and how this affects the secretion of OPE. The data presented here confirm that the moleculeE,E-farnesol accumulates as the cell number is growing within the population. The exogenous addition of this molecule or spent medium to the cultures increased the extracellular activity of OPE 2.5 times. This fact was related not to an increase in microbial biomass or in the expression of the gene coding for OPE but to a marked morphological transition in the cultures. Moreover, the morphological transition also occurred when a high cell density was inoculated into the medium. The results suggest thatE,E-farnesol regulates through QS mechanisms the morphological transition in the dimorphic fungusO. piceaeand that it is associated with a higher extracellular esterase activity. Furthermore, identification and transcriptional analysis of genestup1andcyr1, which are involved in the response, was carried out. Here we report enhanced production of a sterol-esterase/lipase of biotechnological interest by means of QS mechanisms. These results may be useful in increasing the production of secreted enzymes of other dimorphic fungi of biotechnological interest.

Effect of growth conditions on Ophiostoma piceae (Münch) H. & P. Syd. and Ophiostoma floccosum Math-Käärik albino strains culture morphology

Blue stain in Pinus radiata D. Don wood is predominantly caused by Ophiostoma genus fungi. Stained wood causes significant economic losses to forestry and timber industries. Currently, different chemical fungicides are used to prevent the occurrence of blue stain fungi. However, recent reports have questioned the environmental friendliness of these fungicides. For this reason, the use of biological control, in particular with albino strains of the Ophiostoma genus, appears to be an environmentally safe and a technically feasible alternative to work with. The potential field application of this technology would use a formulation containing the albino fungus-the bioactive ingredient- in its yeast like form. However, some Ophiostoma strains cultures present a marked filamentous morphology in liquid culture medium, affecting the efficiency in the production of the bioactive ingredient. The hypothesis of this work presumed the possibility to control the morphology of Ophiostoma cultures, through the modification of growth conditions in liquid medium. The effect of inoculum size, growth temperature and agitation rate on the morphology of PcF2A29 Ophiostoma piceae and FlF1A55 Ophiostoma floccosum albino strains in liquid culture medium, was studied. A 23 factorial design was employed. The results showed that the initial inoculum size had a statistically significant effect on yeast-like growth morphology in both strains, whereas the temperature only produced a significant effect in PcF2A29 O. piceae strain.

Assessing the virulence of ophiostomatoid fungi associated with the pine-infesting weevils to scots pine Pinus sylvestris L. seedlings

The pine-infesting weevils are known to be effective vectors of ophiostomatoid fungi. To understand more about fungal virulence of these fungi, inoculation studies were conducted on Scots pine (<em>Pinus sylvestris</em> L.). Two-year-old seedlings were wound-inoculated with one of eleven ophiostomatoid fungi associated with pine-infesting weevils. After 11 weeks, a darkened lesion, extending from the point of inoculation, was observed in all species, except for <em>Ophiostoma</em> cf. <em>abietinum</em> Marm. &amp; Butin, <em>Ophiostoma quercus</em> (Georgev.) Nannf., and <em>Sporothrix inflata </em>de Hoog. Seedling mortality was observed in seedlings inoculated with <em>Leptographium truncatum</em> (M.J. Wingf. &amp; Marasas) M.J. Wingf., <em>Leptographium lundbergii</em> Lagerb. &amp; Melin<em>,</em> <em>Leptographium procerum</em> (W.B. Kendr.) M.J. Wingf., <em>Grosmannia radiaticola</em> (J.J. Kim, Seifert &amp; G.H. Kim) Zipfel, Z.W. de Beer &amp; M.J. Wingf., <em>Ophiostoma floccosum</em> Math.-Käärik,<em> Ophiostoma minus</em> (Hedgc.) Syd. &amp; P. Syd., and <em>Ophiostoma piliferum </em>(Fr.) Syd. &amp; P. Syd. <em>Ophiostoma minus</em> and <em>L. truncatum</em> caused the largest lesions and sapwood blue-stain in Scots pine. <em>Grosmannia radiaticola, Ophiostoma piceae </em>(Münch) Syd. &amp; P. Syd., <em>O. floccosum</em>,<em> O. piliferum,</em> <em>L. lundbergii</em>,and<em> L. procerum </em>produced significantly smaller lesions and sapwood blue-stain than <em>O. minus</em> and <em>L. truncatum</em>, while<em> O</em>. cf. <em>abietinum</em>, <em>O. quercus</em> and <em>S. inflata</em> did not cause any lesions.