Hydrolysis of PET and bis-(benzoyloxyethyl) terephthalate with a new polyesterase from Penicillium citrinum

A polyethylene terephthalate (PET) model substrate, bis-(benzoyloxyethyl)terephthalate (3PET), was used to screen for micro-organisms producing enzymes hydrolyzing PET. From this screen, a strain growing on 3PET was isolated and identified as Penicillium citrinum. The polyesterase responsible for 3PET and PET hydrolysis was purified to electrophoretic homogeneity. The polyesterase had a molecular weight of 14.1 kDa, and the K_m and K_cat values on 4-nitrophenyl butyrate were 0.57 mM and 0.21 s^-1, respectively. Highest enzyme activities were obtained when P. citrinum was grown on a medium containing cutin, which was hydrolyzed by the polyesterase. Surface hydrolysis of PET with the enzyme lead to an increase in hydrophilicity based on rising height (+5.1 cm) and drop dissipation measurements (55 s). Both from PET and 3PET bis-(2-hydroxyethyl)terephthalate and mono-(2-hydroxyethyl)terephthalate were released, while only low amounts of terephthalic acid were liberated.     

Quinolactacide,a New Quinolone Insecticide from Penicillium citrinum Thom F 1539

By a screening program searching for new pesticides from fungal sources, an insecticidal compound was isolated from Penicillium citrinum F 1539. The compound, named quinolactacide, was novel and showed 88% mortality against green peach aphids (Myzus persicae) at 250 ppm. Its structure was determined by spectroscopic techniques.     

桔青霉中紫杉醇诱导子对红豆杉细胞代谢的影响

在红豆杉细胞培养第２０ｄ时,加入桔青霉紫杉醇诱导子处理５ｄ及１０ｄ后细胞中可溶性蛋白质含量增加,苯丙氨酸解氨酶活性增强,培养基ｐＨ值降低,细胞可溶性蛋白质,过氧化物酶与酯酶同工酶电泳扫描图谱中出现新的谱带为茂本原有个别谱带强度发生变化。     

Quinolinones Alkaloids with AChE Inhibitory Activity from Mangrove Endophytic Fungus Penicillium citrinum YX-002

Acetylcholinesterase (AChE) inhibitory activity-guided studies on the mangrove-derived endophytic fungus Penicillium citrinum YX-002 led to the isolation of nine secondary metabolites, including one new quinolinone derivative, quinolactone A ( 1 ), a pair of epimers quinolactacin C1 ( 2 ) and 3-epi-quinolactacin C1 ( 3 ), together with six known analogs ( 4 – 9 ). Their structures were elucidated based on extensive mass spectrometry (MS) and 1D/2D nuclear magnetic resonance (NMR) spectroscopic analyses, and compared with data in the literature. The absolute configurations of compounds 1 – 3 was determined by combination of electronic circular dichroism (ECD) calculations and X-Ray single crystal diffraction technique using CuK_α radiation. In bioassays, compounds 1 , 4 and 7 showed moderate AChE inhibitory activities with IC₅₀ values of 27.6, 19.4 and 11.2 μmol/L, respectively. The structure-activity relationships (SARs) analysis suggested that the existence of carbonyl group on C-3 and the oxygen atom on the five-membered ring were beneficial to the activity. Molecular docking results showed that compound 7 had a lower affinity interaction energy (−9.3 kcal/mol) with stronger interactions with different sites in AChE activities, which explained its higher activities.     

Statistical Optimization of Alkaline Protease Production from Penicillium citrinum YL-1 Under Solid-State Fermentation

Proteases from halotolerant and halophilic microorganisms were found in traditional Chinese fish sauce. In this study, 30 fungi were isolated from fermented fish sauce in five growth media based on their morphology. However, only one strain, YL-1, which was identified as Penicillium citrinum by internal transcribed spacer (ITS) sequence analysis, can produce alkaline protease. This study is the first to report that a protease-producing fungus strain was isolated and identified in traditional Chinese fish sauce. Furthermore, the culture conditions of alkaline protease production by P. citrinum YL-1 in solid-state fermentation were optimized by response surface methodology. First, three variables including peptone, initial pH, and moisture content were selected by Plackett–Burman design as the significant variables for alkaline protease production. The Box–Behnken design was then adopted to further investigate the interaction effects between the three variables on alkaline protease production and determine the optimal values of the variables. The maximal production (94.30 U/mL) of alkaline protease by P. citrinum YL-1 took place under the optimal conditions of peptone, initial pH, and moisture content (v/w) of 35.5 g/L, 7.73, and 136%, respectively.     

Benzopyran Derivatives and an Aliphatic Compound from a Mangrove Endophytic Fungus Penicillium citrinum QJF‐22

Two new benzopyran derivatives, (2R,4S)‐5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran‐4‐ol and (2S,4R,2′S,4′R)‐4,4′‐oxybis(5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran), and a new aliphatic compound, (3E,5Z,8S,10E)‐8‐hydroxytrideca‐3,5,10,12‐tetraen‐2‐one, together with three known benzopyran derivatives, were obtained from a mangrove endophytic fungus Penicillium citrinum QJF‐22 collected in Hainan island. Their structures were determined by analysis of spectroscopic data and the relative configuration of (2R,4S)‐5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran‐4‐ol was also confirmed by single‐crystal X‐ray diffraction. The absolute configurations of four compounds were established by comparison of ECD spectra to calculations. The configuration of (3E,5Z,8S,10E)‐8‐hydroxytrideca‐3,5,10,12‐tetraen‐2‐one was confirmed by comparison of optical value to the similar compound. The configurations of the compounds (2S,4S)‐5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran‐4‐ol and (2R,4R)‐5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran‐4‐ol were first determined. (3R,4S)‐3,4,8‐Trihydroxy‐3,4‐dihydronaphthalen‐1(2H)‐one exhibited moderate inhibitory effects on LPS‐induced NO production in RAW264.7 cells with IC₅₀ of 44.7 μM, and without cytotoxicity to RAW264.7 cells within 50 μM.     

Synthesis and Degradation of 1-Aminocyclopropane-1-carboxylic Acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M _r 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the K _m for S-adenosyl-L-methionine of 1.74 mM and k _cat of 0.56 s^-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M _r 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a K _m for ACC of 4.8 mM and k _cat of 3.52 s^-1. The presence of 7 mM Cu²⁺ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

The effect of some amino acids on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum Thom 1910

The effect of some amino acids, added to the medium either during inoculation or in the stationary growth phase, on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum VKM FW-800 has been studied. Exogenously added amino acids were mostly utilized in primary metabolism. When added during inoculation, tryptophan and leucine virtually did not influence fungal growth and synthesis of the alkaloids, whereas the addition of isoleucine enhanced the biomass accumulation. When added in the stationary growth phase, tryptophan stimulated the synthesis of both ergot alkaloids and quinocitrinins. Leucine added in the stationary growth phase did not influence the synthesis of ergot alkaloids but inhibited the synthesis of quinocitrinins. Isoleucine inhibited the synthesis of both ergot alkaloids and quinocitrinins irrespective of the time of its addition to the medium.     

橘青霉菌丝球形成条件及其处理废水的应用

探讨了橘青霉 (Penicilliumcitrinum)呈球状体生长的条件和形成的机理。发现在培养液初始pH为 7.0 ,孢子年龄为16～ 2 0天 ,孢子悬液浓度为 10 5个·mL-1,表面活性剂Tween80或TritonX - 10 0质量浓度为 1g/L ,摇床转速为 140r/min的条件下 ,于 2 5℃培养 5天 ,可形成大小适中 ,有一定机械强度 ,光滑均匀的菌丝球。孢子的聚集在菌丝球的形成中起了重要作用。用此菌丝球吸附质量浓度为 5 0mg·L-1的 1 氨基 2 萘酚 4 磺酸水溶液模拟处理染料废水 ,吸附率达到 6 2 .4%。     

A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization

AIMS: The enzymatic hydrolysis of xylan has potential economic and environment-friendly applications. Therefore, attention is focused here on the discovery of new extremophilic xylanase in order to meet the requirements of industry. METHODS AND RESULTS: An extracellular xylanase was purified from the culture filtrate of P. citrinum grown on wheat bran bed in solid substrate fermentation. Single step purification was achieved using hydrophobic interaction chromatography. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular weight of c. 25 kDa and pI of 3.6. Stimulation of the activity by beta mercaptoethanol, dithiotheritol (DTT) and cysteine was observed. Moderately thermostable xylanase showed optimum activity at 50 degrees C at pH 8.5. CONCLUSION: Xylanase purified from P. citrinum was alkaliphilic and moderately thermostable in nature. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work reports for the first time the purification and characterization of a novel endoglucanase free alkaliphilic xylanase from the alkali tolerant fungus Penicillium citrinum. The alkaliphilicity and moderate thermostability of this xylanase may have potential implications in paper and pulp industries.     

Biological control of Botrytis cinerea and plant growth promotion potential by Penicillium citrinum in chickpea (Cicer arietinum L.)

A total of 48 fungi were characterised for their antagonistic potential against Botrytis cinerea causing Botrytis Gray Mold (BGM) disease in chickpea by dual culture and metabolite production assays. The culture filtrate of the most promising isolate, VFI-51, was purified by various chromatographic techniques and identified as ‘citrinin’ by nuclear magnetic resonance and mass spectrometry studies. The efficacy of citrinin was demonstrated to control BGM in chickpea under greenhouse conditions. The sequences of 18S rDNA gene of the VFI-51 matched with Penicillium citrinum in BLAST analysis. The VFI-51 produced siderophore, hydrocyanic acid, indole-3-acetic acid, lipase, protease and β-1,3-glucanase; grew well in NaCl (up to 15%), at pH between 7 and 11 and temperatures between 20°C and 40°C; and was compatible with fungicides bavistin and thiram. Under greenhouse and field conditions, VFI-51 significantly enhanced the nodule number, nodule weight, root and shoot weight and stover and grain yield over the un-inoculated control. In the rhizosphere, VFI-51 also significantly enhanced total N, available P and OC over the un-inoculated control. Scanning electron microscopy analysis revealed that VFI-51 colonised on the roots of chickpea. This study concluded that VFI-51 has the potential for biocontrol of BGM and plant growth promotion in chickpea.     

Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules

A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.     

Hydrolysis of polyethyleneterephthalate by p‐nitrobenzylesterase from Bacillus subtilis

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p‐nitrophenyl acetate and 108 U/mg on p‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The k_cat values decreased with increasing complexity of the substrate from 6 and 8 (s?1) for p‐nitrophenyl‐acetate (4NPA) and p‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s?1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

分离自海藻的青霉属三个中国新记录种

报道了分离自青岛沿海潮间带海藻的青霉属3个中国新记录种:产红青霉Penicillium rubens、苏门答腊青霉P.sumatrense和葡萄生青霉P.viticola,并对其进行了形态学描述和系统发育分析。研究菌株保存于中国海洋大学海洋生物标本室(OUCMB)。     

Purification of a Nuclease from Penicillium citrinum

A nuclease was purified about 1500-fold with a recovery of 20% from an aqueous extract of culture of a pigmentless mutant VI–10–14 of Penicillium citrinum on wheat bran. The purified preparation was homogeneous on the basis of the criteria of ultracentrifugation and disc gel electrophoresis. The preparation was essentially free of 5′-nucleotidase, non-specific phosphomonoesterase, non-specific phosphodiesterase and 3′-monoester forming nuclease. The preparation hydrolyzed phosphodiester bonds in RNA and DNA to yield 5′-mononucleotides, and also the phosphomonoester bond in 2′- and 3′-AMP to yield nucleoside and inorganic phosphate. The enzyme activities toward these substrates were not separated and relative ratio of their specific activities remained constant throughout the purification, suggesting that a single enzyme was responsible for these activities.     

Biocatalytic modification of polyethylene terephthalate fibres by esterases from actinomycete isolates

The modification of polyethylene terephthalate (PET) fibres by extracellular enzymes produced by actinomycetes was investigated. Cultivation of isolates in media containing PET yarn and suberin, a plant polyester composed of aliphatic and aromatic moieties, induced the production of p-nitrophenyl butyrate hydrolyzing enzymes. Incubation of enzyme preparations from the isolates M5, M9 and Thermomonospora fusca KW3b with PET yarn resulted in an increase in the absorbance of the reaction mixtures at 240 nm indicating the release of terephthalic acid or its esters catalyzed by the enzymes. The results of dyeing of enzyme-treated PET fabrics with a reactive dye (CI Reactive Red 2) indicated an increase in hydroxyl groups at the fibre surfaces as a result of the enzyme treatment.     

Biocatalytic modification of polyethylene terephthalate fibres by esterases from actinomycete isolates

The modification of polyethylene terephthalate (PET) fibres by extracellular enzymes produced by actinomycetes was investigated. Cultivation of isolates in media containing PET yarn and suberin, a plant polyester composed of aliphatic and aromatic moieties, induced the production of p-nitrophenyl butyrate hydrolyzing enzymes. Incubation of enzyme preparations from the isolates M5, M9 and Thermomonospora fusca KW3b with PET yarn resulted in an increase in the absorbance of the reaction mixtures at 240 nm indicating the release of terephthalic acid or its esters catalyzed by the enzymes. The results of dyeing of enzyme-treated PET fabrics with a reactive dye (CI Reactive Red 2) indicated an increase in hydroxyl groups at the fibre surfaces as a result of the enzyme treatment.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

由扩展青霉(Penicillium expansum)PF868产生脂肪酶催化油脂水解的研究

研究了由扩展青霉（Ｐｅｎｉｃｉｌｉｕｍｅｘｐａｎｓｕｍ）ＰＦ８６８产生脂肪酶催化水解三种油脂（橄榄油、豆油、鱼油）的影响因素与工艺条件,其中包括：水解时间、温度、ｐＨ、酶量、油水比及添加剂,并用气相色谱对产品脂肪酸进行了分析鉴定,初步分析其催化水解的脂肪酸的特异性