Nuclease Rsn from Rhizopus stolonifer: specificity and mode of action

Nuclease Rsn from Rhizopus stolonifer catalyzes the hydrolysis of ss- and dsDNA in a ratio of approximately 2:1. Time course of 3' and 5' terminal analysis of the hydrolytic products of ss- and dsDNA showed that nuclease Rsn does not show any strict base preference and cleaves DNA in a non-specific manner. Moreover, separation of the hydrolytic products of ss- and dsDNA in the presence of Mg2+, Mn2+ or Co2+ showed the predominance of tetra-, tri-, and dinucleotides followed by mononucleotides, suggesting an endo mode of action.     

Optimization of medium for extracellular nuclease formation from Rhizopus stolonifer

A strain of Rhizopus stolonifer produced a high activity of extracellular DNAase when grown on YPG (yeast extract peptone glucose) medium. The source of peptone had a marked effect on the enzyme yield and only one peptone (i.e. from Sarabhai M. Chemicals Ltd, India) supported enzyme production. Maximum enzyme activity (88 U/ml) was obtained after 4 days' growth under submerged conditions in YPG medium containing 100 M Mn²⁺, Co²⁺ or Mg²⁺, and glucose as the sole carbon source. The unpurified enzyme was optimally active at pH 7.5 and 45°C. It had a higher activity with sonicated and heat-denatured DNA than native DNA.     

Genetic variability in the species Rhizopus stolonifer, assessed by random amplified polymorphic DNA analysis

Rhizopus stolonifer is an important post-harvest pathogenic fungus. Recent taxonomic findings based on morphological and growth characteristics led to a dramatic reduction in the number of accepted species within the genus. The aim of this study was to examine this situation with molecular markers. Twenty-nine R. stolonifer strains isolated from various locations and substrates were characterized by random amplified polymorphic DNA (RAPD) analysis. The numerical analysis of the RAPD data revealed four main clusters with extremely high dissimilarity values, but only low or moderate variability was observed within these groups. These results suggest a high genetic heterogeneity in the case of R. stolonifer: isolates of R. stolonifer var. stolonifer, R. stolonifer var. reflexus and R. niveus displayed species-level genetic distances, which gives rise to considerations that they might be separate species.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Biotransformation of mestanolone and 17-methyl-1-testosterone by Rhizopus stolonifer

Abstract

Microbial transformation of mestanolone (1) using the plant pathogenic fungus, Rhizopus stolonifer, resulted in the production of two known metabolites, identified as 11α-hydroxymestanolone (3) and 6α-hydroxymestanolone (4). Transformation of 17-methyl-1-testosterone (2) by R. stolonifer yielded two known metabolites, methandrostenolone (5) and 11α,17β- dihydroxy-androsta-1,4-diene-3-one (6). These transformations included α-hydroxylations at C-11 and C-6, dehydrogenation at C-4, androsta and a demethylation at C-17 positions. Structures of transformed products were determined using spectroscopic techniques.     

Purification of the extracellular pectinolytic enzyme from the fungus Rhizopus oryzae NBRC 4707

The pectinolytic enzyme from the solid-state culture of Rhizopus oryzae NBRC 4707 was purified to homogeneity by column chromatography on CM-Toyopearl 650 M and hydroxylapatite. The molecular weight of the enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 31,000 and was reduced to 29,700 after treatment with endoglycosidase H. Maximal activity was observed near pH 4.5 at 45°C. The enzyme was shown to be endopolygalacturonase, as judged from the formation of oligogalacturonides as its reaction products. The addition of purified enzyme, as expected, enhanced the formation of lactic acid and ethanol in potato pulp grown with R. oryzae.     

Both mating types in the heterothallic fungus Ophiostoma quercus contain MAT1-1 and MAT1-2 genes

In heterothallic Ascomycota, two opposite but distinct mating types control all sexual processes. Using mating crosses, mating types were assigned to ten isolates of the heterothallic fungal species Ophiostoma quercus. Primers were subsequently designed to target the MAT1-1-1, MAT1-1-3 (of the mating type 1 idiomorph), and MAT1-2-1 (of the mating type 2 idiomorph) genes in these isolates. Results showed that all isolates contained the full gene sequence for the MAT1-2-1 gene. In addition, fragments of the MAT1-1-1 and MAT1-1-3 genes were sequenced from all isolates. These results were unexpected, as each isolate from a heterothallic species would typically contain only one of the two possible MAT idiomorphs.     

Investigation of the importance of the C-2 oxygen function in the transformation of stemodin analogues by Rhizopus oryzae ATCC 11145

A new stemodinoside, stemodin-alpha-L-arabinofuranoside (5), was isolated from the plant Stemodia maritima. Incubation of stemodin (2) with Rhizopus oryzae ATCC 11145 gave 2 alpha,7 beta,13(S)-trihydroxystemodane (17) and 2 alpha,3 beta,13(S),16 alpha-tetrahydroxystemodane (18) whilst stemodinone (8) afforded 6 alpha,13(S)-dihydroxystemodan-2-one (19). The bioconversion of 2 beta,13(S)-dihydroxystemodane (10) by the fungus yielded 2 beta,7 beta,13(S)-trihydroxystemodane (20) whereas stemod-12-en-2-one (9) provided 7 beta,17-dihydroxystemod-12-en-2-one (21). The results provide useful information about the relationship between the functional groups of the substrates and their potential for bioconversion.     

Investigation of the importance of the C-2 and C-13 oxygen functions in the transformation of stemodin analogues by Rhizopus oryzae ATCC 11145

Incubation of 2alpha,13(R)-dihydroxystemodane (3) with Rhizopus oryzae ATCC 11145 gave 2alpha,7beta,13(R)-trihydroxystemodane (11) while biotransformation of 13(R)-hydroxystemodan-2-one (5) yielded 6alpha,13(R)-dihydroxystemodan-2-one (12) and 7beta,13(R)-dihydroxystemodan-2-one (13). Bioconversion of 2beta,13(R)-dihydroxystemodane (7) with Rhizopus afforded 2beta,7,13(R)-trihydroxystemodane (14). The results complement data from our previous work and provide more information about the effect of functional groups of stemodane substrates on the site of hydroxylation.     

Liposome-mediated mycelial transformation of filamentous fungi

Liposome-mediated transformation is common for cells with no cell wall, but has very limited usage in cells with walls, such as bacteria, fungi, and plants. In this study, we developed a procedure to introduce DNA into mycelium of filamentous fungi, Rhizopus nigricans LH 21 and Pleurotus ostreatus TD 300, by liposome-mediation but with no protoplast preparation. The DNA was transformed into R. nigricans via plasmid pEGFP-C1 and into P. ostreatus via 7.2 kb linear DNA. The mycelia were ground in 0.6 M mannitol without any grinding aids or glass powder for 15 min to make mycelial fragments suspension; the suspension was mixed with a mixture of the DNA and Lipofectamine 2000, and placed on ice for 30 min; 100 μL of the transformation solution was plated on potato dextrose agar (PDA) plate and cultivated at 28 °C for transformant screening. The plasmid and the linear DNA were confirmed to be integrated into the host chromosome, proving the success of transformation. The transformation efficiencies were similar to those of electroporation-mediated protoplast transformation (EMPT) of R. nigricans or PEG/CaCl₂-mediated protoplast transformation (PMT) of P. ostreatus, respectively. The results showed that our procedure was effective, fast, and simple transformation method for filamentous fungi.     

Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production

The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.     

Biotransformation of adrenosterone by filamentous fungus, Cunninghamella elegans

Microbial transformation of adrenosterone (1) by suspended-cell cultures of the filamentous fungus Cunninghamella elegans resulted in the production of five metabolites 2-6, which were identified as 9alpha-hydroxyadrenosterone (2), 11-ketotestosterone (3), 6beta-hydroxyadrenosterone (4), 9alpha-hydroxy-11-ketotestosterone (5), and 6beta-hydroxy-11-ketotestosterone (6). Structures of new metabolites 2, 5, and 6 were established by single-crystal X-ray diffraction analysis.     

Phytotoxins from the fungus Malbranchea aurantiaca

Bioassay-directed fractionation of an ethyl acetate extract from cultures of the fungus Malbranchea aurantiaca led to the isolation of two phytotoxic compounds, namely, 1-hydroxy-2-oxoeremophil-1(10),7(11),8(9)-trien-12(8)-olide (1) and penicillic acid (2). The structure of 1 was established by spectroscopic and X-ray crystallographic analyses. Metabolites 1 and 2 caused significant inhibition of radicle growth of Amaranthus hypochondriacus with IC(50) values 6.57 and 3.86 microM, respectively. In addition, 1 inhibited activation of the calmodulin-dependent enzyme cAMP phosphodiesterase (IC(50)=10.2 microM).     

Influence of some environmental factors on Rhizopus nigricans submerged growth in the form of pellets

Morphological changes of a steroid transforming filamentous fungus Rhizopus nigricans were studied by altering submerged growth conditions at inoculum sizes previously determined to favor pelleted growth. Beside the main classification between pelleted and clumpy growth forms, the size, concentration and structure of pellets were characterized at different cultivating temperature, initial pH value, medium composition, additives, and aeration conditions. Initial pH below 4 and above 7, the presence of Ca²⁺ and Tween-80 gave rise to the clumpy growth, otherwise pelleted growth prevailed. Among tested factors the pellet size was mainly influenced by the inoculum size and the presence of baffles and Ca²⁺ in cultivation medium. The formation of smooth pellets, prerequisite for further application in the process of steroid biotransformation, resulted in cultivations at lower temperature, high agitation rates in shaken cultures without baffles and at high nitrogen concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130±102 and 1200±97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35% of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels.     

Characterisation of the extracellular polysaccharides produced by isolates of the fungus Acremonium

Solid state (13)C NMR studies of the extracellular glucans from the fungi Acremonium persicinum C38 (QM107a) and Acremonium sp. strain C106 indicated a backbone of (1-->3)-beta-linked glucosyl residues with single (1-->6)-beta-linked glucosyl side branches for both glucans. Analyses of enzymatic digestion products suggested that the average branching frequency for the A. persicinum glucan (66.7% branched) was much higher than that of the Acremonium sp. strain C106 glucan (28.6% branched). The solid state (13)C NMR spectra also indicated that both glucans are amorphous polymers with no crystalline regions, and the individual chains are probably arranged as triple helices.     

碳阻遏因子CRE对匍枝根霉产纤维素酶调控作用的研究

为研究匍枝根霉(Rhizopus stolonifer)TP-02中碳代谢阻遏因子CRE对其产纤维素酶的调控特性,从其基因组DNA中扩增得到CRE全长基因,并利用重叠PCR(overlapping PCR)技术将潮霉素B抗性基因(hygB)插入其中,既破坏cre正常转录又提供了筛选标记。通过电转化将pUCm-T-cre-hygB导入匍枝根霉萌发孢子,经抗性筛选得到△CRE突变株。利用RT-qPCR对此突变株纤维素酶基因转录水平进行研究,发现 eg、bg、cbh1和cbh2 的转录均有所提高,分别为48.75%、26%、5.6%和38.6%。同时,△CRE突变株的纤维素酶在表达水平上也均高于原菌,其中内切葡聚糖苷酶酶活提高了58.62%。CRE的破坏在一定程度上减弱了碳阻遏效应,其对纤维素酶的调控具有特异性。其中,对内切酶的调控最为显著。此外,△CRE突变株在3%糖浓度下仍高产纤维素酶,这为后续酶系优化及产业化生产提供了一定的依据。     

Production of phytase under solid-state fermentation using Rhizopus oryzae: novel strain improvement approach and studies on purification and characterization

Present study introduces linseed oil cake as a novel substrate for phytase production by Rhizopus oryzae. Statistical approach was employed to optimize various medium components under solid state fermentation (SSF). An overall 8.41-fold increase in phytase production was achieved at the optimum concentrations (w/w, mannitol, 2.05%; ammonium sulfate, 2.84% and phosphate, 0.38%). Further enhancement by 59% was observed due to a novel strain improvement approach. Purified phytase (～34 kDa) showed optimal temperature of 45 °C, dual pH optima at 1.5 and 5.5 and possesses high catalytic efficiency (2.38×10(6) M(-1) s(-1)). Characterization study demonstrates the phytase as highly thermostable and resistant to proteolysis, heavy metal ions, etc. Furthermore, an improved HPLC method was introduced to confirm the ability of phytase to degrade phytic acid completely and was found to be an efficient method.     

基于响应面法优化匍枝根霉TP-02液态发酵纤维素酶条件

利用Plackett-Burman设计法(Plackett-Burman,PB),对影响根霉TP-02液态发酵产纤维素酶的8个因子进行了筛选,结果表明,影响该菌发酵产纤维素酶的主要因子为麸皮与稻草的比例、槐糖、Tween 80。利用最陡爬坡试验逼近最大响应区域,在此基础上,采用响应面法(ResponseSurface Methodology,RSM)对这3个因子的影响进行研究,得出纤维素酶产量的数学模型,通过对二次多项回归方程求解,得到3个因子的最优用量:麸皮稻草比例为:3.7:1,槐糖量为:0.62%,Tween 80为0.68 g/L,在优化后的条件下培养96 h,纤维素酶滤纸酶活可达到8.13 IU/mL比优化前提高了38.97%。     

Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus

Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C_16:0, C_16:1, C_18:0, C_18:1, C_18:2, C_18:3, C_20:0 and C_20:1. At high concentrations spore germination was inhibited by C_7:0, C_8:0, C_9:0, C_10:0, C_12:0, C_18:2 and C_18:3 and hyphal growth was merely retarded by C_5:0, C_6:0, C_6:2, C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_20:0 and C_20:1. The presence of C_15:0 at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C_16:0 and C_18-20 fatty acids. Low concentrations of C_5:0, C_6:0, C_6:2 and C_7:0 enhanced sporulation. Fatty acids C_5-12 as well as C_18:3, C_20:0 and C_20:1 decreased the ability of fungal colonies to infect G. mellonella while C_16:1 elevated it thus suggesting that C_16:1 may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C_6:0, C_6:2, C_7:0, C_9:0, C_12:0, C_16:1, C_18:2, C_18:3, C_20:0 and C_20:1; other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.