Characterization of the distribution of glucose oxidase in Penicillium sp. CBS 120262 and Aspergillus niger NRRL-3 cultures and its effect on integrated product recovery

Glucose oxidase (GO) is an important industrial enzyme typically purified from Penicillium and Aspergillus sp. As GO distribution within the cultures influences process design for maximal product recovery, distribution of GO activity in Penicillium sp. CBS 120262 and Aspergillus niger NRRL-3, during mid-exponential and stationary phases, is compared. On progression from mid-exponential to stationary phase, the percentage GO activity in the cytoplasm decreased 1.6- and 1.3-fold in Penicillium sp. and A. niger respectively. In Penicillium sp., a concomitant 1.8- and 1.9-fold decrease in the percentage GO activity in the cell envelope and slime mucilage respectively, translated into a 2.0-fold increase in the extracellular fluid. In A. niger, decreasing cytoplasmic GO activity was accompanied by 1.3-fold increases in the cell envelope and slime mucilage, with a 1.3-fold decrease in the extracellular fluid. Similar trends were observed in specific GO activities. As final GO activity recovered is governed by the purification program, recovery from the extracellular fluid plus cell extract or from the extracellular fluid only were compared through simulating processes of varying complexity. A critical yield for each purification stage was identified above which recovery from the extracellular fluid plus cell extract exceeded that from extracellular fluid alone. These results highlight the influence of microorganism, harvest time and efficiency of downstream process on GO activity delivered. In the systems studied, Penicillium sp. is the organism of choice and should be harvested during stationary phase. The purification process chosen should be informed by both enzyme distribution and individual purification stages yields.     

来源于Bacillus sp. AR9 D-海因酶半理性设计的研究

D-对羟基苯甘氨酸是一种重要的精细化工品，在制药行业具有广泛的应用前景。酶法是生产D-对羟基苯甘氨酸的主要手段，但由于缺乏高催化效率的酶而限制了D-对羟基苯甘氨酸的生产。为了提高来自Bacillus sp. AR9的D-海因酶（HYD）的催化效率，进而提高D-对羟基苯甘氨酸的产量，对HYD的底物结合通道进行分析，选取底物通道瓶颈处的氨基酸进行饱和突变和筛选，以提高HYD的催化效率。结果显示，突变体F159S、F159A和F65V的活性相较于野生型HYD分别提高了51%、40%和17%，通过对突变体F65V、F159S和双位点突变F65V/F159S的酶动力学研究发现，突变体的Km值基本与野生型HYD相似，而kcat是野生型HYD的1.3、1.9和2.0倍，最终双位点突变F65V/F159S的催化效率kcat/Km是野生型HYD的2.4倍。高催化效率突变体的获得，以及对突变体动力学的分析，对酶法制备D-对羟基苯甘氨酸具有重要的研究意义和应用价值。     

Studies on a morphologically distinct colchicine-resistance variant of Entamoeba sp.

Colchicine has a temperature-dependent cytotoxic effect on Entamoeba sp. (Laredo isolate) that is most apparent when the drug is applied during the initiation of cultures at a concentration of 7.5 mM or higher. Continued transfer of cultures in medium containing progressively increasing concentrations of colchicine has resulted in a variant that grows prolifically in the presence of colchicine (7.5 mM) with a generation time comparable to that of the parent stock, Comparison of a number of parameters of the 2 variants revealed that colchicine resistance was accompanied by a change in cell shape, a reduced membrane permeability, which could partially be overcome by the addition of dimethyl sulfoxide (DMSO), and a reduced tolerance to osmotic stress. However, the parent strain and resistant variant were equally susceptible to cycloheximide and puromycin suggesting that the acquired colchicine resistance may not be explained on the basis of an entirely unspecific generalized reduced ability for drug uptake. Colchicine resistance and altered structure were found to be stable over a long period of time. The possible interdependence of these 2 parameters and their relation to cell motility in Entamoeba sp. are discussed.     

Biotransformation of geraniol, nerol and citral by sporulated surface cultures of Aspergillus niger and Penicillium sp

The biotransformation of geraniol, nerol and citral by Aspergillus niger was studied. A comparison was made between submerged liquid, sporulated surface cultures and spore suspensions. This bioconversion was also carried out with surface cultures of Penicillium sp. The main bioconversion products obtained from geraniol and nerol by liquid cultures of A. niger were linalool and alpha-terpineol. Linalool, alpha-terpineol and limonene were the main products obtained from nerol and citral by sporulated surface cultures, whereas geraniol was converted predominantly to linalool, also resulting in higher yields. Bioconversion of nerol with Penicillium chrysogenum yielded mainly alpha-terpineol and some unidentified compounds. With P. rugulosum the major bioconversion product from nerol and citral was linalool. The bioconversion of nerol to alpha-terpineol and linalool by spore suspensions of A. niger was also investigated. Finally the biotransformation with sporulated surface cultures was also monitored by solid phase microextraction (SPME). It was found that SPME is a very fast and efficient screening technique for biotransformation experiments.     

Isolation,Identification and Screening of Manganese Solubilizing Fungi From Low-Grade Manganese Ore Deposits

The present investigation reports the isolation, molecular identification and screening of manganese (Mn) solubilizing fungal strains from low-grade Mn mine tailings. Six morphologically distinct Mn solubilizing fungal strains were isolated on MnO₂-supplemented agar plates with Mn concentration of 0.1% (w/v). The biochemical characterization of the isolated fungal strains was carried out. The molecular identification by internal transcribed spacer (ITS) sequencing identified the strains as Aspergillus terreus, Aspergillus oryzae, Penicillium sp., Penicillium sp., Penicillium daleae and Penicillium sp. with GenBank accession numbers KP309809, KP309810, KP309811, KP309812, KP309813 and KP309814, respectively. The ability of the isolated fungal strains to tolerate and solubilize Mn was investigated by subculturing them on Mn-supplemented plates with concentration ranging from 0.1 to 0.5% (w/v). Mn solubilizing ability of the fungal isolates is possibly due to the mycelia production of biogenerated organic acids such as oxalic acid, citric acid, maleic acid and gluconic acid as revealed by ion chromatography. Our investigation signifies the role of fungi in biotransformation of insoluble Mn oxide.     

Studies on a Morphologically Distinct Colchicine-Resistant Variant of Entamoeba sp.*

Colchicine has a temperature-dependent cytotoxic effect on Entamoeba sp. (Laredo isolate) that is most apparent when the drug is applied during the initiation of cultures at a concentration of 7.5 mM or higher. Continued transfer of cultures in medium containing progressively increasing concentrations of colchicine has resulted in a variant that grows prolifically in the presence of colchicine (7.5 mM) with a generation time comparable to that of the parent stock. Comparison of a number of parameters of the 2 variants revealed that colchicine resistance was accompanied by a change in cell shape, a reduced membrane permeability, which could partially be overcome by the addition of dimethyl sulfoxide (DMSO), and a reduced tolerance to osmotic stress. However, the parent strain and resistant variant were equally susceptible to cycloheximide and puromycin suggesting that the acquired colchicine resistance may not be explained on the basis of an entirely unspecific generalized reduced ability for drug uptake. Colchicine resistance and altered structure were found to be stable over a long period of time. The possible interdependence of these 2 parameters and their relation to cell motility in Entamoeba sp. are discussed.     

Biosorption of Ni, Cr and Cd by metal tolerant Aspergillus niger and Penicillium sp. using single and multi-metal solution

Fungi including Aspergillus and Penicillium, resistant to Ni2+, Cd2+, and Cr6+ were isolated from soil receiving long-term application of municipal wastewater mix with untreated industrial effluents of Aligarh, India. Metal tolerance in term of minimum inhibitory concentration (MIC) was 125-550 microg/ml for Cd, 300-850 microg/ml for Ni and 300-600 microg/ml for Cr against test fungi. Two isolates, Aspergillus niger and Penicillium sp. were tested for their Cr, Ni and Cd biosorption potential using alkali treated, dried and powdered mycelium. Biosorption experiment was conducted in 100 ml of solution at three initial metal concentrations i.e., 2, 4 and 6 mM with contact time (18 hr) and pretreated fungal biomass (0.1g) at 25 degrees C. Biosorption of all metals was found higher at 4 mM initial metal concentration as compared to biosorption at 2 and 6 mM concentrations. At 4 mM initial metal concentration, chromium biosorption was 18.05 and 19.3 mg/g of Aspergillus and Penicillium biomasses, respectively. Similarly, biosorption of Cd and Ni ions was also maximum at 4 mM initial metal concentration by Aspergillus (19.4 mg/g for Cd and 25.05 mg/g of biomass for Ni) and Penicillium (18.6 mg/g for Cd and 17.9 mg/g of biomass for Ni). In general, biosorption of metal was influenced by initial metal concentration and type of the test fungi. The results indicated that fungi of metal contaminated soil have high level of metal tolerance and biosorption properties.     

Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus

In a prior study, we identified seven clinical isolates of an Aspergillus sp. that were slow to sporulate in multiple media and demonstrated decreased in vitro susceptibilities to multiple antifungals, including amphotericin B, itraconazole, voriconazole, and caspofungin. These isolates were initially considered to be variants of Aspergillus fumigatus because of differences in mitochondrial cytochrome b sequences and unique randomly amplified polymorphic DNA PCR patterns (S. A. Balajee, M. Weaver, A. Imhof, J. Gribskov, and K. A. Marr, Antimicrob. Agents Chemother. 48: 1197-1203, 2004). The present study was performed to clarify the taxonomic status of these organisms by phylogenetic analyses based on multilocus sequence typing of five genes (the beta-tubulin gene, the rodlet A gene, the salt-responsive gene, the mitochondrial cytochrome b gene, and the internal transcribed spacer regions). Results revealed that four of the seven variant isolates clustered together in a clade very distant from A. fumigatus and distinct from other members of the A. fumigatus group. This new clade, consisting of four members, was monophyletic with strong bootstrap support when the protein-encoding regions were analyzed, indicating a new species status under the phylogenetic species concept. Phenotype studies revealed that the variant isolate has smaller conidial heads with diminutive vesicles compared to A. fumigatus and is not able to survive at 48 degrees C. Our findings suggest the presence of a previously unrecognized, potentially drug-resistant Aspergillus species that we designate A. lentulus.     

Arsenate transport and reduction in the hyper-tolerant fungus Aspergillus sp. P37

Aspergillus sp. P37 is able to grow at arsenate concentrations of 0.2 M--more than 20-fold higher than that withstood by reference microorganisms such Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. This paper examines the transport of arsenate and phosphate and the reduction of arsenate in Aspergillus sp. P37. These properties were compared with the corresponding properties of the archetype strain Aspergillus nidulans TS1. Both uptake and efflux of arsenate were inhibited by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, suggesting that the transport system(s) is(are) membrane-potential dependent. As uptake of arsenate and phosphate are higher in Aspergillus sp. P37 than in A. nidulans, the increase in arsenate resistance cannot be accounted for by a change in uptake. Cells of both strains loaded with arsenic slowly released the oxyanion. Speciation of the arsenic in the medium showed an enhanced level of arsenate reduction in Aspergillus sp. P37. These data suggest that increased arsenate reduction is at least in part responsible for the hyper-tolerant phenotype of this fungus.     

Motility comparisons of two shape-distinctiveEntamoeba variants

Summary The motility ofEntamoeba sp. (Laredo isolate, LA) is compared to a colchicine-resistant variant (LACR) derived from it and known to exhibit physiological and morphological differences. Both variants possess contractile elements which in glycerinated cell models were activated by Mg²⁺-ATP. Short-term experiments indicated that whereas both variants are insensitive to colchicine, treatment with cytochalasin B induces a change in cell shape and loss of adhesiveness in LA cells leaving LACR relatively unaffected. Quantitative analysis of time-lapse cinematographic recordings revealed that the mean relative surface area change of LA cells was 411% per minute with a mean pseudopod extension rate of 103 m per minute. In contrast, the respective values for LACR cells were 112% per minute und 32 m per minute. Cytochalasin B treatment reduced the relative surface area change of LA cells by approximately 18-fold with a corresponding reduction in LACR cells of only 1.6-fold. Similarly, cytochalasin B inhibited pseudopod extension by 34 and 1.8-fold in LA and LACR cells respectively. Thus, we have demonstrated a clear difference in both pattern and rate of locomotion of the two variants and have shown that the colchicine resistant variant has also become relatively insensitive to treatment with CB.     

Host specificity of Eupenicillium ochrosalmoneum, E. cinnamopurpureum and two Penicillium species associated with the conidial heads of Aspergillus

The genus Penicillium comprises species that mostly colonize plant matter. However early reports suggest that several species are capable of parasitizing Aspergillus and sporulating on the conidial heads of the host. More recently Eupenicillium ochrosalmoneum and E. cinnamopurpureum, both with Penicillium anamorphs, have been observed sporulating on the heads of Aspergillus species belonging to section Flavi during the colonization of peanut seeds. Little is known about the host specificity underlying these Aspergillus-Penicillium associations. In this study Aspergillus species representing nine taxonomic sections were paired in culture with E. ochrosalmoneum, E. cinnamopurpureum and two unnamed Penicillium species. Eupenicillium ochrosalmoneum, E. cinnamopurpureum and Penicillium sp. 1 sporulated predominantly on the heads of section Flavi species. In contrast Penicillium sp. 2 was restricted to the heads of section Nigri species. All species spread across Aspergillus colonies by means of aerial hyphae that grew from head to head. Additional studies are required to clarify whether Eupenicillium and Penicillium species are parasitic or simply epibiotic on their hosts.     

Role of arginine 59 in the gamma-class carbonic anhydrases.

The functional role of the highly conserved active site Arg 59 in the prototype of the gamma-class carbonic anhydrase Cam (carbonic anhydrase from Methanosarcina thermophila) was investigated. Variants (R59A, -C, -E, -H, -K, -M, and -Q) were prepared by site-directed mutagenesis and characterized by size exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, and stopped-flow kinetic analyses. CD spectra indicated similar secondary structures for the wild type and the R59A and -K variants, independent of nondenaturing concentrations of guanidine hydrochloride (GdnHCl). SEC indicated that all variants purified as homotrimers like the wild type. SEC also revealed that the R59A and -K variants unfolded at > or = 1.5 M GdnHCl, compared to 3.0 M GdnHCl for the wild type. These results indicate that Arg 59 contributes to the thermodynamic stability of the Cam trimer. The R59K variant had k(cat) and k(cat)/K(m) values that were 8 and 5% of the wild-type values, respectively, while all other variants had k(cat) and k(cat)/K(m) values 10-100-fold lower than those of the wild type. The R59A, -C, -E, -M, and -Q variants exhibited 4-63-fold increases in k(cat) and 9-120-fold increases in k(cat)/K(m) upon addition of 100 mM GdnHCl, with the largest increases observed for the R59A variant, which was comparable to the R59K variant. The kinetic results indicate that a positive charge at position 59 is essential for the CO(2) hydration step of the overall catalytic mechanism.     

Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.     

Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) by denitrifying Pseudomonas sp. strain FA1

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 +/- 0.1 nmol h(-1) mg of cell biomass(-1) and 11.5 +/- 0.4 nmol h(-1) mg of protein(-1), respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO(2)(-)), 1.5 molecules of nitrous oxide (N(2)O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.     

A <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. dextranase mutant pool with improved thermostability and activity

Random mutagenesis was used to create a library of chimeric dextranase (dex1) genes. A plate-screening protocol was developed with improved thermostability as a selection criterion. The mutant library was screened for active dextranase variants by observing clearing zones on dextran-blue agar plates at 50°C after exposure to 68°C for 2 h, a temperature regime at which wild-type activity was abolished. A number of potentially improved variants were identified by this strategy, five of which were further characterised. DNA sequencing revealed ten nucleotide substitutions, ranging from one to four per variant. Thermal inactivation studies showed reduced (2.9-fold) thermostability for one variant and similar thermostability for a second variant, but confirmed improved thermostability for three mutants with 2.3- (28.9 min) to 6.9-fold (86.6 min) increases in half-lives at 62°C compared to that of the wild-type enzyme (12.6 min). Using a 10-min assay, apparent temperature optima of the variants were similar to that of the wild type (T _opt 60°C). However, one of these variants had increased enzyme activity. Therefore, the first-generation dextranase mutant pool obtained in this study has sufficient molecular diversity for further improvements in both thermostability and activity through recombination (gene shuffling).     

Chromosomally located gene fusions constructed in Acinetobacter sp. ADP1 for the detection of salicylate

Acinetobacter sp. ADP1 is a common soil-associated bacterium with high natural competency, allowing it to efficiently integrate foreign DNA fragments into its chromosome. This property was exploited to engineer salicylate-inducible luxCDABE and green fluorescent protein (GFP) variants of Acinetobacter sp. ADP1. Specifically, Acinetobacter sp. ADPWH_lux displayed the higher sensitivity when comparing the two variants (minimum detection c. 0.5-1 microM salicylate) and a faster turnover of the lux marker gene, making it suitable for whole-cell luminescence assays of salicylate concentration. In contrast, the longer maturation and turnover times of the GFP protein make the Acinetobacter sp. ADPWH_gfp variant more suited to applications involving whole-cell imaging of the presence of salicylate. The sensitivity of the luxCDABE variant was demonstrated by assaying salicylate production in naphthalene-degrading cultures. Assays using ADPWH_lux specifically mapped the kinetics of salicylate production from naphthalene and were similar to that observed by high-performance liquid chromatography (HPLC) data. However, ADPWH_lux exhibited the higher sensitivity, when compared with HPLC, for detecting salicylate production during the first 24 h of naphthalene metabolism. These data demonstrate that the engineered Acinetobacter variants have significant potential for salicylate detection strategies in laboratory and field studies, especially in scenarios where genetic stability of the construct is required for in situ monitoring.     

Efficient biotransformation for preparation of pharmaceutically active ginsenoside Compound K by Penicillium oxalicum sp. 68

Pathogenic fungus Penicillium oxalicum sp. 68 was screened from soil and identified by ITS sequencing. The strain was found to be able to transform protopanaxadiol-type ginsenosides to produce a series of bioactive metabolites. Glycosidase from the culture of P. oxalicum sp. 68 was partially purified with a simple two-step procedure consisting of DEAE-cellulose chromatography and ammonium sulfate precipitation. Bioactive ginsenoside Compound K was prepared selectively and efficiently by biotransformation of ginsenosides Rb1, Rb2, Rc and Rd using the partially purified glycosidase. The optimal conditions for transforming Rb1 into Compound K were pH 4.0, 55 °C and 0.5 mg mL^?1 Rb1. The sole product is Compound K and the maximum yield reached 87.7 % (molar ratio). The transformation pathways of Rb1, Rb2, Rc and Rd are Rb1→Rd→F2→Compound K, Rb2→CO→CY→Compound K, Rc→Mb→Mc→Compound K and Rd→F2→Compound K, respectively. This biotransformation method showed great potential for preparing minor bioactive ginsenosides, especially Compound K, in the pharmaceutical industry because of its high specificity and favorable environmental compatibility.     

Morphology and ultrastructure of a Penicillium sp. grown on n-hexadecane or peptone.

The morphology and ultrastructure of a Penicillium sp. grown on n-hexadecane or on peptone in shake-culture were compared using transmission and scanning electron microscopy. The fungus grew as hollow mycelial balls surrounding individual hydrocarbon droplets on n-hexadecane and as solid mycelial balls on peptone. A dense layer of fungal mycelium that showed irregular forms, fusion, and increase in hyphal size formed at the hydrocarbon-water interface. Inclusions were present in the hexadecane-grown fungus that were absent when the Penicillium sp. was grown on peptone. Problems of fixation made it difficult to differentiate detailed changes in the cytoplasm when the fungus was examined with the transmission electron microscope.     

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.