Penicillium chrysogenum glucose oxidase -- a study on its antifungal effects

AIMS: Purification and characterization of the high molecular mass Candida albicans-killing protein secreted by Penicillium chrysogenum. METHODS AND RESULTS: The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (M(r)) = 155 000, subunit structure alpha(2) with M(r,alpha) = 76 000, isoelectric point (pI) = 5.4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1.0 mg ml(-1)) prevented oxidative cell injuries triggered by 0.004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0.004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX-catalase system was likely to replace the primary inhibition exerted by H(2)O(2). CONCLUSIONS: Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent.     

Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products

EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.     

A novel approach for screening immunogenic proteins in Penicillium marneffei using the DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus

Using serum from guinea-pigs immunized with a DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus to screen a cDNA library of A. fumigatus, we cloned a novel immunogenic 57-kDa protein in A. fumigatus. We also cloned its 55-kDa homologue in Penicillium marneffei, which was possibly related to amino acid biosynthesis and metabolism, with homologues present only in the subphylum Pezizomycotina of Ascomycota. The recombinant 55-kDa protein of P. marneffei reacted strongly with guinea-pig serum immunized with P. marneffei and with the sera of patients with P. marneffei infection. A similar approach could be applied to immunogenic protein screening in other microorganisms for serological diagnosis, epidemiological studies and the study of vaccines.     

The influence of culture conditions on the biosynthesis of secondary metabolites by Penicillium verrucosum Dierck

A Brazilian strain of Penicillium verrucosum was cultivated under different conditions in a two-step process, in order to verify the influence of nutrients, and of time periods of pre-fermentative and fermentative steps on the biosynthesis of metabolites. Extracellular and intracellular extracts were obtained from each culture in the four different production media used. Chemical profiles of the extracts were obtained by HPLC. Extract trypanocidal activities against trypomastigote forms of Trypanosoma cruzi were evaluated. The time period of incubation in the pre-fermentative and fermentative media, as well as the different nutrients tested, qualitatively and quantitatively modified the production of secondary metabolites by P. verrucosum, and the extract trypanocidal activities.     

产胞外布雷菲德菌素A青霉发酵条件的研究

通过正交设计试验,优化了青霉(Penicillium)属真菌SHZK-15产胞外布雷菲德菌素(B refeld in-A,简称BFA)的液体发酵条件。最佳的BFA发酵条件为:培养基含马铃薯200g(煮汁),葡萄糖20g,(NH4)2SO44.0g,KH2PO41.0g,MgSO4.7H2O 2.0g,CaCO35.0g,pH自然,瓶装量为100mL/250 mL,培养温度为28℃,转速为120 r/m in,培养时间为7d,BFA的最高产量可达151.6mg/L。     

Expression and homology modeling of sterol 14alpha-demethylase from Penicillium digitatum

Green mold of citrus, caused by Penicillium digitatum, is the most serious postharvest disease of citrus. Sterol 14alpha-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in biological kingdoms and is a prime target of antifungal drugs. To exploit novel 14alpha-demethylase inhibitor (DMI) fungicides, DNA and total RNA were isolated from P. digitatum. The CYP51 of P. digitatum was cloned and expressed in Escherichia coli, yielding recombinant protein with a molecular weight of c. 59 kDa. The P. digitatum CYP51 protein (PdCYP51) was purified and polyclonal antibodies were prepared. Compared with the sequence of P. digitatum PD5 in GenBank, there were four mutated nucleotides which resulted in four mutated amino acids. The three-dimensional (3D) model of P. digitatum CYP51 was established based on structure template of 1e9x.pdb and diniconazole was docked into the active site by FlexX. According to spectral data, it is suggested that the purified soluble protein had high affinity with diniconazole, a potent inhibitor of CYP51 reaction in fungi. At the same time, these spectral data suggested that the 3D model and the docking model were reasonable, which we hope can be used to provide a virtual screening of novel DMI drugs.     

四个嗜热真菌中国新记录种

报道四个嗜热真菌中国新记录种:嗜热子囊菌光孢变种Thermoascus aurantiacus var.levisporus;埃默森篮状菌Talaromyces emersonii;杜邦青霉Penicillium dupontii及丝衣霉状拟青霉Paecilomyces byssochlamydioides,对其进行了描述和讨论。研究标本保存在山东农业大学植物病理学标本室(HSAUP)。     

K55R与ep8叠加突变对扩展青霉脂肪酶热稳定性的改善

利用重叠延伸PCR对扩展青霉脂肪酶（PEL）基因进行体外定点突变,构建了K55R与随机突变体ep8叠加突变的重组质粒pAO815-ep8-K55R。将该质粒电转化引入毕赤酵母（Pichia pastoris）GS115,进行异源表达。实验结果表明：该叠加突变体在毕赤酵母中获得了活性表达,得到表达产物脂肪酶PEL-ep8-K55R-GS。其表达量为508u/mL,分别约为野生型脂肪酶PEL-GS（627u/mL）的81％,随机突变脂肪酶PEL-ep8-GS（924u/mL）的55%;其比活力为2309.1u/mg,与随机突变脂肪酶PEL-ep8-GS和野生型脂肪酶PEL-GS的相仿。叠加突变脂肪酶PEL-ep8-K55R-GS的最适作用温度为37℃,与野生型脂肪酶PEL-GS和随机突变脂肪酶PEL-ep8-GS一致;其T_m值为41.0℃,比野生型脂肪酶PEL-GS提高了2.3℃,比随机突变脂肪酶PEL-ep8-GS提高了0.8℃。表明叠加突变脂肪酶PEL-ep8-K55R-GS的热稳定性有了进一步的提高。     

Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina

AIMS: The aims of this work were to evaluate the mycoflora and to identify the species of Aspergillus with the potential to produce ochratoxin A (OA) from different wine grape varieties from Mendoza, Argentina. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. METHODS AND RESULTS: Fifty samples of wine grapes were obtained from a winery of Mendoza province, Argentina. The surface-disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18). Alternaria, Aspergillus and Penicillium were identified at species level. OA production was tested in 63 strains belonging to section Nigri. Alternaria genus was the most frequent (80% of the samples) followed by Aspergillus (70%). Alternaria alternata was the only specie identified from the Alternaria genus, followed by A. niger var. niger, A. flavus among others. From Penicillium genus, P. crysogenum was the most frequent specie. From 63 strains of Aspergillus section Nigri, 41.3% were OA producers. The levels of produced toxin ranged from 2 to 24.5 ng ml-1 of culture medium. CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in this substrate suggests that they may be an important source of OA in grapes from tropical and subtropical zones. Therefore, the industry should work further to diminish the growth of these fungi and mycotoxins formation in grapes, with the aim to reduce OA content in wine products. SIGNIFICANCE AND IMPACT OF THE STUDY: The wine grape contamination with A. alternata and Aspergillus section Nigri was significant.