Effect of environmental conditions on extracellular lipases production and fungal morphology from Aspergillus niger MYA 135

Under the current assay conditions, lipase production in mineral medium was only detected in the presence of vegetable oils, reaching the highest specific activity with olive oil. In this way, effect of different environmental conditions on fungal morphology and olive oil‐induced extracellular lipases production from Aspergillus niger MYA 135 was studied. It was observed that addition of 1.0 g l^–1 FeCl₃to the medium encouraged filamentous growth and increased the specific activity 6.6 fold after 4 days of incubation compared to the control. However, major novelty of this study was the satisfactory production of an acidic lipase at initial pH 3 of the culture medium (1.74 ± 0.06 mU μg^–1), since its potencial applications in food and pharmaceutical industry are highly promising. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Production of an extracellular thermohalophilic lipase from a moderately halophilic bacterium, Salinivibrio sp. strain SA-2

Fifty strains of moderately halophilic bacteria were isolated from various salty environments in Iran. A strain designated as SA-2 was shown to be the best producer of extracellular lipase and was selected for further studies. Biochemical and physiological characterization along with 16S rDNA sequence analysis placed SA-2 in the genus Salinivibrio. The optimum salt, pH, temperature and aeration for enzyme production were 0.1 M KCl, pH 8, 35 degrees C and 150 rpm, respectively. The enzyme production was synchronized bacterial growth and reached a maximum level during the early-stationary phase in the basal medium containing 1 M NaCl. Triacylglycerols enhanced lipase production, while carbohydrates had inhibitory effects on it. The maximum lipase activity was obtained at pH 7.5, 50 degrees C and CaCl(2) concentration of 0.01 M. The enzyme was stable at pH range of 7.5-8 and retained 90% of its activity at 80 degrees C for 30 min. Different concentrations of NaNO(3), Na(2)SO(4), KCl and NaCl had no affect on lipase stability for 3 h. These results suggest that the lipase secreted by Salinivibrio sp. strain SA-2 is industrially important from the perspective of its tolerance to a broad temperature range, its moderate thermoactivity and its high tolerance to a wide range of salt concentrations (0-3 M NaCl).     

Mycelium-bound lipase production from Aspergillus niger MYA 135, and its potential applications for the transesterification of ethanol

The potential biotechnological applications of both constitutive and inducible lipase sources from Aspergillus niger MYA 135 were evaluated. To this end, the effect of environmental conditions on mycelium-bound lipase production from this strain was studied, when cultured either in the absence or presence of 2% olive oil. It was previously reported that mycelium-bound lipase from Aspergillus niger MYA 135 possess high stability in reaction mixtures containing ethanol; which could be especially important for their use in biodiesel synthesis. In this connection, the performance of the lipase sources produced in the transesterification of ethanol using p-nitrophenyl palmitate as acyl donor was also explored. Under our assay conditions, hydrolytic and synthetic activity of the mycelia produced in the absence or presence of olive oil were not highly correlated. While the hydrolytic activity was strongly increased by the addition of lipid to the culture medium, the best performance in the transesterification reactions of ethanol were associated with mycelia produced in absence of olive oil. Interestingly, the supplementation of the culture medium with Fe(+3) increased the transesterification activity by 71%, as compared to the activity previously reported for this strain. Therefore, the constitutive lipase sources from Aspergillus niger MYA 135 are considered to be promising for industrial biodiesel-fuel production.     

Transesterification of used edible and non-edible oils to alkyl esters by Aspergillus sp. as a whole cell catalyst

Aspergillus sp. (MTCC 5436), isolated from contaminated clarified butter was used as a whole cell catalyst for transesterification of oils from different sources. The strain was observed to be tolerant and grow in 90% oil as carbon source. Oils of Jathropa, karanj and spent cottonseed were used as carbon sources in the study. The product, alkyl ester, was characterized and quantified using (1) H-NMR. The strain was observed to facilitate transesterification in an oil:minimal medium with the ratio of 70:30 resulting in a 98% conversion of oil to ethyl esters within 48 h at 28 °C and 120 rpm. The physico-chemical characteristics of the ethyl ester (>98%) at 70% oil as carbon source were similar to the standards specified for biodiesel as per standards of American Society for Testing and Materials (ASTM) and Bureau of Indian Standards (BIS), India.     

The application of PCR for the isolation of a lipase gene from the genomic DNA of an Antarctic microfungus

We successfully isolated a lipase gene (designated lipPA) directly from the genomic DNA of an Antarctic isolate of Penicillium allii using PCR and a suite of degenerate primers specifically designed to target two conserved regions of fungal lipase genes. We applied the biolistic transformation system to successfully integrate the lipPA gene into a heterologous fungal host, Trichoderma reesei, one of the most powerful secretors of extracellular proteins, and induced the transformant to secrete an active lipase into the growth medium. The recombinant lipase had a temperature optimum of 25 °C at pH 7.9 and retained greater than 50% of the maximum activity from 10 °C to 35 °C and over a pH range from 4.0 to 8.5.Communicated by U. Kück     

Isolation and characterization of the nuclease O gene (nucO) from Aspergillus oryzae

Nuclease O in the mycelia of Aspergillus oryzae has been purified 55-fold by successive steps of chromatography from the filtrate of the autolyzate. The molecular mass of nuclease O was 32 kDa, as estimated by SDS polyacrylamide-gel electrophoresis. The nuclease O gene (nucO) encoding this enzyme was cloned and sequenced. The open reading frame is interrupted by four introns with conserved splice sites and contains 328 amino-acid residues of the mature enzyme. A. nidulans transformants obtained by introduction of the cloned nucO gene produced 2.5-times as much nuclease O as the wild-type strain, showing that the cloned DNA fragment encodes nuclease O. Received: 6 March / 15 May 1996     

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus

Summary Wa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.The sequence reported in this paper has been submitted to the EMBL/GenBank through DDBJ (accession no, X52525).     

Micro‐RNA‐155‐mediated control of heme oxygenase 1 (HO‐1) is required for restoring adaptively tolerant CD4+ T‐cell function in rodents

T cells chronically stimulated by a persistent antigen often become dysfunctional and lose effector functions and proliferative capacity. To identify the importance of micro‐RNA‐155 (miR‐155) in this phenomenon, we analyzed mouse miR‐155‐deficient CD4⁺ T cells in a model where the chronic exposure to a systemic antigen led to T‐cell functional unresponsiveness. We found that miR‐155 was required for restoring function of T cells after programmed death receptor 1 blockade. Heme oxygenase 1 (HO‐1) was identified as a specific target of miR‐155 and inhibition of HO‐1 activity restored the expansion and tissue migration capacity of miR‐155^?/? CD4⁺ T cells. Moreover, miR‐155‐mediated control of HO‐1 expression in CD4⁺ T cells was shown to sustain in vivo antigen‐specific expansion and IL‐2 production. Thus, our data identify HO‐1 regulation as a mechanism by which miR‐155 promotes T‐cell‐driven inflammation.     

Microbial production of docosahexaenoic acid by a low temperature‐adaptive strain Thraustochytriidae sp. Z105: screening and optimization

As an alternative source in addition to fish oil, microbial production of docosahexaenoic acid has been recieved more and more attentions owing to their culture advantage. A unicellular eukaryotic microbe with high DHA production and capable of low temperature‐adaptive growth was isolated from seawater and identified as Thraustochytriidae sp. Z105. The siginificant effect of temperature on cell growth and DHA synthesis by the strain was revealed. It could grow and produce DHA even at 4 °C, but hardly grow above 35 °C. Low temperature (15–25 °C) was favorable for formation of biomass, lipids and DHA, but DHA synthesis was completely blocked above 30 °C. Conditions for high level DHA production by Thraustochytriidae sp. Z105 in flask culture were optimized as follows: medium containing glucose 80 g/l, yeast extract 5.0 g/l, K₂HPO₄ · 3 H₂O 1.0 g/l, MgSO₄ · 7 H₂O 0.5 g/l, seawater crystal 20 g/l, pH 6.0, liquid volume 30 ml/250 ml, temperature 20 °C, agitation speed of 200 r/min, and culture for 120 h. Under the optimal conditions, biomass of 16.72 g/l, total lipids of 5.35 g/l, DHA yield of 1.71 g/l (accounting for 32% of the total lipids) were achieved, respectively. In flask cluture level, the DHA productivity of Thraustochytriidae sp. Z105 was higher than most reported results, which suggested the wild type strain was a potential superior candidate for industrialization of DHA production. Moreover, the strain is an unique and valuable resource for investigation of the low temperature adaptive mechanism related to DHA synthesis. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Optimization of different process variables for the production of an indolizidine alkaloid, swainsonine from Metarhizium anisopliae

Swainsonine is a polyhydroxylated indolizidine alkaloid having anticancer, antimetastatic, antiproliferative and immunomodulatory activities and also potential therapeutic applications against AIDS. In the present study, ten isolates of M. anisopliae were screened and enzyme assayed for the production of swainsonine in different media (Complex oatmeal, Czapekdox media with and without lysine (8% w/v) and Sabouraud dextrose broth (SDB)). Among these strains, ARSEF 1724 (UM8) was found to produce highest amount of swainsonine (1.34 μg/l) after 72 h of incubation under shake flask conditions at 180 rpm and 28 °C in complex oatmeal media. In order to maximize the yield of swainsonine the media composition including macro and micronutrients were optimized. The process variables including the chemical factors like carbon sources, nitrogen sources of both organic and inorganic nature and pH with constant inoculum size (1 × 10⁸ spores/ml) were screened using classical one‐factor‐at‐a‐time (OFAT) approach to find their optimum levels. The present study shows that the nutrient requirement is specific for each strain of Metarhizium. Oatmeal extract (6%) was found to be the best supporting media along with nitrogen source, glucose (2%) as best carbon source and pH (～5) as the best for swainsonine production. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L

A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5′-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger. Received: 10 August / 13 October 1998     

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae

Summary Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T₂ gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T₂ revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T₂ activity than wild-type strains.     

Comparison of the EUCAST-AFST broth dilution method with the CLSI reference broth dilution method (M38-A) for susceptibility testing of posaconazole and voriconazole against Aspergillus spp.

The susceptibilities of 40 clinical isolates of Aspergillus spp. (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus) were determined for posaconazole and voriconazole by the CLSI M38-A and EUCAST-AFST broth dilution methods. Where a discrepancy was observed between the methods, the EUCAST method tended to give higher MIC values. Overall, the level of agreement was 92.5% and the intra-class correlation coefficient was > 0.90.