Biotransformation of imperatorin by Penicillium janthinellum. Anti-osteoporosis activities of its metabolites

Imperatorin (IMP) is a major constituent of many herbal medicines and possesses anti-osteoporosis activity. The present research work aimed to study the biotransformation processes of IMP and evaluated the anti-osteoporosis activity of the transformed metabolites. Among 18 strains of filamentous fungi screened, Penicillium janthinellum AS 3.510 exhibited good capability to metabolise IMP to the new derivatives. Ten transformed products were isolated and purified, and their structures were identified accurately based on spectroscopic data. Eight metabolites (2–8 and 10) were novel and previously unreported. The major biotransformation reactions involved hydroxylation of the prenyloxy side-chain and the lactone ring-opening reaction of furocoumarin skeleton. In addition, anti-osteoporosis activities of all products (1–10) were evaluated using MC3T3-E1 cells. The results showed that products 5 and 8 had the best bioactivities in increasing MC3T3-E1 cell growth. These products could be used in future therapeutic regimens for treating osteoporosis.     

Inhibition of Penicillium expansum by an oxidative treatment

Several oxidizing compounds such as sodium hypochlorite (NaClO) and hydrogen peroxide (H₂O₂) are used to control postharvest decay in fresh fruit due to their antimicrobial effects. Here, we applied these compounds in vitro, in the presence of CuSO₄, against Penicillium expansum, causal agent of apple blue mold. MICs were 50 mg L⁻¹ and 400 mmol L⁻¹ for NaClO and H₂O₂, respectively, when these compounds were individually applied to conidia suspensions during 2 min. A combined oxidative treatment (OT) consisting on an incubation with 1 mg L⁻¹ NaClO and 200 mmol L⁻¹ H₂O₂, in the presence of 6 mmol L⁻¹ CuSO₄, inhibited growth, conidial germination and fungal infectivity on apple. The fractional inhibitory concentration index for the interaction between NaClO and H₂O₂ in the OT was 0.52 indicating a synergistic effect of the oxidizing compounds. These results suggest that the OT could be an interesting alternative for apple diseases postharvest control.     

圆弧青霉碱性脂肪酶在毕赤酵母中的高效表达

目的在毕赤酵母(Pichia pastoris)中高效表达圆弧青霉碱性脂肪酶(Alkaline lipase,LipⅠ)。方法采用RT-PCR法从圆弧青霉PG37中扩增lipⅠ基因的cDNA片段,克隆入表达质粒pPIC9K中,构建重组表达质粒pPIC9K-lipⅠ,转化入毕赤酵母GS115,筛选高拷贝重组子GS115/lipⅠ,甲醇诱导表达,并对诱导表达条件进行初步优化。结果 GS115/lipⅠ在培养基初始pH6.0,甲醇添加量1.0%的BMMY培养基中,于30℃诱导96h,发酵液上清中LipⅠ的活性为10.5U/ml。在培养基初始pH9.0,甲醇添加量1.0%的BMMY培养基中,24℃诱导120h,GS115/lipⅠ发酵液上清中LipⅠ的活性最高,达407U/ml。结论在毕赤酵母GS115中高效表达了具有活性的圆弧青霉LipⅠ。     

红树林土壤纤维素酶产生菌筛选及产酶条件研究

采用CMC唯一碳源平板法和内切葡聚糖酶、外切葡聚糖酶和β-葡萄糖苷酶等3种酶平板鉴别法从海南红树林土壤中分离到109个有阳性信号的菌株,经发酵产酶复筛选出一株产纤维素酶活相对较高的真菌HBZ003。经鉴定该菌为产紫青霉(Penicillium purpurogenum)。通过发酵产酶条件优化,获得最佳培养基组成为:麸皮8 g/L,CMC 2 g/L,(NH4)2SO43 g/L,KNO32 g/L,KH2PO43 g/L,NaCl 6 g/L,CaCl20.5 g/L;发酵条件为250mL三角瓶中装培养液100mL,在pH4.0、30℃,160 r/min条件下振荡培养5 d,测得发酵液中CMCase和FPA分别为16.04U和4.08 U。     

The effects of the combination of Pichia membranefaciens and BTH on controlling of blue mould decay caused by Penicillium expansum in peach fruit

The feasibility of 0.2 g l⁻¹ benzo-thiadiazole-7-carbothioic acid S-methyl ester (BTH) to improve the efficacy of Pichia membranefaciens in controlling postharvest blue mould decay in peach fruit was investigated. Our results showed that biocontrol activity of P. membranefaciens against blue mould caused by Penicillium expansum in peach fruit could be enhanced by addition of 0.2 g l⁻¹ BTH. The combination of P. membranefaciens and BTH resulted in a more effective control of blue mould than individual treatment of P. membranefaciens or BTH alone. The combined treatment had a synergistic effect on the induction of superoxide dismutase, catalase, ascorbate peroxidase, chitinase and β-1,3-glucanase activities, which induced stronger disease resistance in fruit than BTH or yeast alone, and resulted in a lower lesion diameter and disease incidence of blue mould decay in peaches. Furthermore, the combined treatment did not impair the quality parameters including fruit firmness and contents of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 °C. These results suggested that the use of BTH may be an effective method to improve the biological activity of P. membranefaciens.     

Enzymatic properties and transglycosylation of α-galactosidase from Penicillium oxalicum SO

Penicillium oxalicum SO α-galactosidase demonstrated weak hydrolysing activity but a high rate of transglycosylation in the reaction with melibiose, where the major product was 6-α-galactosyl melibiose. The transfer ratio was 83.6% and was maintained over a long reaction time of 80 h. The molecular weight was estimated to be 124,000 by SDS–PAGE. The optimal pH was ∼3 and a stable pH, with a range of 2.4–9.5, was found. The optimal temperature was ∼60 °C and the activity was stable below 60 °C. With respect to acceptor specificity, mono-alcohols, sugar alcohols and sugars were poor acceptors, but the di-alcohol ethylene glycol and the tri-alcohol glycerin were good acceptors. The percentage of transglycosylation to glycerin increased up to 41.7%, as that to melibiose decreased, with the initial glycerin concentration of 40%. The production of α-d-galactosylglycerol was 293 mg for each gram of melibiose used by the enzymatic reaction.     

Bioassay-guided isolation and identification of antifungal components from propolis against Penicillium italicum

The present study was aimed at identification of antifungal components against Penicillium italicum from Chinese propolis with bioassay-guided fractionation technique. Propolis ethanolic extract (PEE) was separated and purified by liquid–liquid extraction and thin layer chromatography (TLC) and the most active band was subjected to HPLC–MS/MS to identify the antifungal compounds. The results showed PEE and its fractions had strong antifungal activity against P. italicum. Among the fractions of PEE partitioned by petroleum ether, ethyl acetate, n-butanol and water, ethyl acetate fraction (E-Fr) exhibited the most effective activity against P. italicum. Further bioautographic TLC assay showed Band I, with Rf value of 0.70, had an inhibitive zone, which showed the strongest antifungal activity and completely inhibited the growth of P.italicum at 200 mg/L. Bioactive components found in Band I were further identified as pinobanksin, pinocembrine, chrysin and galangin. This study exhibited Chinese propolis and its main flavonoids was potential natural alternatives for the control of citrus blue mould caused by P.italicum.     

Comparative study of flavonoid and scoparone accumulation in different Citrus species and their susceptibility to Penicillium digitatum

Fungi of the genus Penicillium are responsible for substantial post-harvest losses in Citrus fruits. The results obtained following artificial inoculation of Citrus fruits with Penicillium digitatum showed that the degree of fungal development depended on the Citrus species. Thus, the mature fruit of Citrus paradisi were more susceptible to this fungus than the mature fruit of Citrus limon, Citrus sinensis,Citrus clementina Hort. ex Tan., and Citrus unshiu (mak) Marc. The results point to an inverse correlation between the degree of susceptibility of Citrus species to this fungus and the flavanone content – hesperidin in C. sinensis, C. clementina Hort. ex Tan. and C. unshiu (mak) Marc.; naringin in C. paradisi and the flavanone hesperidin and the flavone diosmin in C. limon. Thus, in C. sinensis,C. clementina Hort. ex Tan and C. unshiu (mak) Marc. the highest levels of the polymethoxyflavones, sinensetin, tangeretin, heptamethoxyflavone and nobiletin, were observed in the least susceptible varieties and viceversa. In the case of C. paradisi, no significant differences were detected in the polymethoxyflavone levels between varieties, while in C. limon, polymethoxyflavones were hardly detectable. The production of scoparone was observed in all the species and varieties studied after inoculation with the fungus, especially in C. limon fruits. Based on the evidence, it seems that flavanones, flavones, polymethoxyflavones (phytoanticipins) and scoparone (a phytoalexin) may well be involved in the defence mechanisms of Citrus fruits against P. digitatum. Depending on the Citrus species in question, the relative participation of one group of secondary compounds or another may vary.     

产壳聚糖酶的海洋青霉菌筛选及其寡营养发酵的探究

从海边海螺、贝壳及其附着物的微生物中筛选出产壳聚糖酶的青霉菌。以此菌为出发菌,以降低发酵产酶成本为目的,采用不同的发酵温度,分别添加不同诱导物进行发酵产酶,探讨温度、诱导物对菌种生长、酶活力的影响。实验结果表明:低聚糖诱导产酶效果最好,室温25℃进行壳聚糖酶发酵,普通培养基酶活力最高达3.57U/mL。海水寡营养发酵产酶的适宜培养基配方为(g/L):蛋白胨9g、葡萄糖10g的海水培养基,酶活力可以达到3.16U/mL,初步实现利用海水进行寡营养发酵产酶,该菌株具有工业应用潜力。     

Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens

An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M_r) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M_r of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50 °C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.