Diversity of plant oil seed-associated fungi isolated from seven oil-bearing seeds and their potential for the production of lipolytic enzymes.

Commercial oil-yielding seeds (castor, coconut, neem, peanut, pongamia, rubber and sesame) were collected from different places in the state of Tamil Nadu (India) from which 1279 endophytic fungi were isolated. The oil-bearing seeds exhibited rich fungal diversity. High Shannon-Index H' was observed with pongamia seeds (2.847) while a low Index occurred for coconut kernel-associated mycoflora (1.018). Maximum Colonization Frequency (%) was observed for Lasiodiplodia theobromae (176). Dominance Index (expressed in terms of the Simpson's Index D) was high (0.581) for coconut kernel-associated fungi, and low for pongamia seed-borne fungi. Species Richness (Chao) of the fungal isolates was high (47.09) in the case of neem seeds, and low (16.6) for peanut seeds. All 1279 fungal isolates were screened for lipolytic activity employing a zymogram method using Tween-20 in agar. Forty isolates showed strong lipolytic activity, and were morphologically identified as belonging to 19 taxa (Alternaria, Aspergillus, Chalaropsis, Cladosporium, Colletotrichum, Curvularia, Drechslera, Fusarium, Lasiodiplodia, Mucor, Penicillium, Pestalotiopsis, Phoma, Phomopsis, Phyllosticta, Rhizopus, Sclerotinia, Stachybotrys and Trichoderma). These isolates also exhibited amylolytic, proteolytic and cellulolytic activities. Five fungal isolates (Aspergillus niger, Chalaropsis thielavioides, Colletotrichum gloeosporioides, Lasiodiplodia theobromae and Phoma glomerata) exhibited highest lipase activities, and the best producer was Lasiodiplodia theobromae (108 U/mL), which was characterized by genomic sequence analysis of the ITS region of 18S rDNA.

Microbial demethylation of lignin: Evidence of enzymes participating in the removal of methyl/methoxyl groups.

Lignin is an abundant natural plant aromatic biopolymer containing various functional groups that can be exploited for activating lignin for potential commercial applications. Applications are hindered due to the presence of a high content of methyl/methoxyl groups that affects reactiveness. Various chemical and enzymatic approaches have been investigated to increase the functionality in transforming lignin. Among these is demethylation/demethoxylation, which increases the potential numbers of vicinal hydroxyl groups for applications as phenol-formaldehyde resins. Although the chemical route to lignin demethylation is well-studied, the biological route is still poorly explored. Bacteria and fungi have the ability to demethylate lignin and lignin-related compounds. Considering that appropriate microorganisms possess the biochemical machinery to demethylate lignin by cleaving O-methyl groups liberating methanol, and modify lignin by increasing the vicinal diol content that allows lignin to substitute for phenol in organic polymer syntheses. Certain bacteria through the actions of specific O-demethylases can modify various lignin-related compounds generating vicinal diols and liberating methanol or formaldehyde as end-products. The enzymes include: cytochrome P450-aryl-O-demethylase, monooxygenase, veratrate 3-O-demethylase, DDVA O-demethylase (LigX; lignin-related biphenyl 5,5'-dehydrodivanillate (DDVA)), vanillate O-demethylase, syringate O-demethylase, and tetrahydrofolate-dependent-O-demethylase. Although, the fungal counterparts have not been investigated in depth as in bacteria, O-demethylases, nevertheless, have been reported in demethylating various lignin substrates providing evidence of a fungal enzyme system. Few fungi appear to have the ability to secrete O-demethylases. The fungi can mediate lignin demethylation enzymatically (laccase, lignin peroxidase, manganese peroxidase, O-demethylase), or non-enzymatically in brown-rot fungi through the Fenton reaction. This review discusses details on the aspects of microbial (bacterial and fungal) demethylation of lignins and lignin-model compounds and provides evidence of enzymes identified as specific O-demethylases involved in demethylation.

A rapid method to detect and estimate the activity of the enzyme, alcohol oxidase by the use of two chemical complexes - acetylacetone (3,5-diacetyl-1,4-dihydrolutidine) and acetylacetanilide (3,5-di-N-phenylacetyl-1,4-dihydrolutidine).

A rapid and sensitive method has been devised in order to detect and estimate the synthesis of the enzyme alcohol oxidase (AOX) by fungi, by way of the use of two chemical complexes, namely, acetylacetone (3,5-diacetyl-1,4-dihydrolutidine) and acetylacetanilide (3,5-di-N-phenylacetyl-1,4-dihydrolutidine). This method involves the use of the AOX enzyme that could specifically oxidize methanol, giving rise to equimolar equivalents each of formaldehyde (HCHO) and hydrogen peroxide (H2O2) as the end products. Further, the formaldehyde, thus produced was allowed to interact with the neutral solutions of acetylacetone and the ammonium salt, gradually developing a yellow color, owing to the synthesis and release of 3,5-diacetyl-1,4-dihydrolutidine (yellow product; λ = 420 nm; λex/em = 390/470 nm) and the product, so generated was quantified spectrophotometrically by measureing its absorbance at 412 nm. In another set up, the amount of formaldehyde produced as a sequel to the oxidation of methanol by the AOX enzyme was determined by allowing it to react with the acetylacetanilide reagent, after which the volume of the fluorescent product - 3,5-di-N-phenylacetyl-1,4-dihydrolutidine (colorless product; λex/em = 390/470 nm) that was generated was estimated by measuring its emission at 460 nm (excitation wavelength at 360 nm) in a spectrophotometer. Of the various substrates tested, a commercial source of the AOX enzyme appreciably oxidizes methanol, thereby generating formaldehyde, and further reacts with acetylacetone, to give rise to a bright yellow complex, displaying a maximum activity of 1402 U/mL. Determination of the AOX activity by the use of acetylacetone and acetylacetanilide could serve as a viable alternative to the conventional alcohol oxidase-peroxidase-2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (AOX-POD-ABTS) based method. In view of this, this method appears to be invaluable for application at the various food, pharmaceutical, fuel, biosensor, biorefinery, biopolymer, biomaterial, platform chemical, and biodiesel industries.

Enzymatic Kraft lignin demethylation and fungal O-demethylases like vanillate-O-demethylase and syringate O-demethylase catalyzed catechol-Fe3+ complexation method.

The ability of enzymatic Kraft Lignin (KL) demethylation was determined using catechol and ferric ion coordination (catechol-Fe3+ complexes) by reduction of Fe3+ to Fe2+ and formation of mono, bis- and/or tris-catechol-Fe3+ complexes has been investigated to identify enzyme that can strip-off O-methyl groups from lignin such as O-demethylase. To detect fungal demethylation and release of catechol-like structures, these were demonstrated using catechol, gallic acid and caffeic acid as standard model compounds to forms mono, bis- and/or tris-catechol-Fe3+ complexes. The catechol-Fe3+ complexes formation controlled by pH via the deprotonation of the catechol hydroxyls was investigated at pH 2.5, 8.0 and 10.0 and demonstrated that catechol formed mono, bis- and/or tris-catechol-Fe3+ complexes, and showed maximum absorbance at 547 nm. Lignin demethylation (O-demethylase) and formation of pyrocatecholic structures was detected using Aspergillus sp. and Galerina autumnalis culture filtrates as the enzyme source. The produced aromatic vicinal diol groups in lignin model compounds (LMCs) and KL were determined using different catecholic-binding reagents with the influence of H2O2, along with 4-antiaminopyrine reagent, was analyzed by the following: i) Fe3+-catechol complexation method, ii) HNO2 method, iii) FAS (Ferric Ammonium-Sulfate) method, iv) Ti(III)-NTA (Titanium (III)- Nitrilotriacetate) method for hydrolytic zone formation. Among the tested methods showing lytic zone formation was Fe3+-catechol complexation. The LMCs and KL treated using Aspergillus sp. culture filtrate showed maximum Fe3+-catechol complexes with 3-methoxy catechol (91 µmol/mL), o-vanillin (44 µmol/mL) and KL (100 µmol/mL). In addition, Galerina autumnalis culture filtrate showed demethylation of vanillin (48 µmol/mL), 3-methoxy catechol (82 µmol/mL), o-vanillin, (33 µmol/mL), 3 4-dimethoxybenzyl alcohol (49 µmol/mL) and KL (41 µmol/mL). The results suggest that lignin demethylation (O-demethylases) activity that strip-off methyl groups in LMCs and KL and produced vicinal diols that covalently bind with Fe3+ to form Fe3+-catechol complexes. The new Fe3+-catechol complexation method has the ability to characterize pyrocatechol and galloyl structures in chemically or biologically modified lignins and to detect O-demethylase activity.

The Purification and Characterization of Lipases from Lasiodiplodia theobromae, and Their Immobilization and Use for Biodiesel Production from Coconut Oil.

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)-purified 25.41-fold, recovery of 47.1%-and lipase B (32,000 Da)-purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca2+, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5-10.0 and 20-80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.