Production of Phenolic Compounds by Aspergillus S-4 in Sake Cake Medium and Identification of Terrein

High performance liquid chromatography (HPLC) analysis of the culture filtrate of an isolated mold, Aspergillus terreus S-4, showed a larger peak of a terrein-like substance than those of gallic acid and protocatechuic acid. The chemical structure of this substance was investigated and identified as terrein, 4,5-dihydroxy-3-propenyl- 2-cyclopenten-1-one. We also examined some culture conditions for the production of terrein using a sake cake medium for strain S-4.     

Upstream and Downstream Processing of Lovastatin by Aspergillus terreus

The present study describes the enhanced production and purification of lovastatin by Aspergillus terreus in submerged batch fermentation. The enhancement of lovastatin production from A. terreus was attempted by random mutagenesis using ultraviolet radiations and nitrous acid. UV mutants exhibited increased efficiency for lovastatin production as compared with nitrous acid mutants. Among all the mutants developed, A. terreus UV-4 was found to be the hyper producer of lovastatin. This mutant gave 3.5-fold higher lovastatin production than the wild culture of A. terreus NRRL 265. Various cultural conditions were also optimized for hyper-producing mutant strain. 5 % glucose as carbon source, 1.5 % corn steep liquor as nitrogen source, initial pH value of 6, 120 h of incubation period, and 28 °C of incubation temperature were found as best parameters for higher lovastatin production in shake flasks. Production of lovastatin by wild and mutant strains of A. terreus was also scaled up to laboratory scale fermentor. The fermentation process was conducted at 28 °C, 200 rpm agitation, and 1vvm air flow rate without pH control. After the optimization of cultural conditions in 250 ml Erlenmeyer flasks and scaling up to laboratory scale fermentor, the mutant A. terreus UV-4 gave eightfold higher lovastatin production (3249.95 μg/ml) than its production by wild strain in shake flasks. Purification of lovastatin was carried out by solvent extraction method which yielded 977.1 mg/l of lovastatin with 98.99 % chromatographic purity and 26.76 % recovery. The crystal structure of lovastatin was determined using X-ray diffraction analysis which is first ever reported.     

Vitamins A & D inhibit the growth of mycobacteria in radiometric culture

Background

The role of vitamins in the combat of disease is usually conceptualized as acting by modulating the immune response of an infected, eukaryotic host. We hypothesized that some vitamins may directly influence the growth of prokaryotes, particularly mycobacteria.

Methods

The effect of four fat-soluble vitamins was studied in radiometric Bactec® culture. The vitamins were A (including a precursor and three metabolites,) D, E and K. We evaluated eight strains of three mycobacterial species (four of M. avium subspecies paratuberculosis (MAP), two of M. avium and two of M. tb. complex).

Principal Findings

Vitamins A and D cause dose-dependent inhibition of all three mycobacterial species studied. Vitamin A is consistently more inhibitory than vitamin D. The vitamin A precursor, β-carotene, is not inhibitory, whereas three vitamin A metabolites cause inhibition. Vitamin K has no effect. Vitamin E causes negligible inhibition in a single strain.

Significance

We show that vitamin A, its metabolites Retinyl acetate, Retinoic acid and 13-cis Retinoic acid and vitamin D directly inhibit mycobacterial growth in culture. These data are compatible with the hypothesis that complementing the immune response of multicellular organisms, vitamins A and D may have heretofore unproven, unrecognized, independent and probable synergistic, direct antimycobacterial inhibitory activity.     

Emerging biotechnologies for production of itaconic acid and its applications as a platform chemical

Recently, itaconic acid (IA), an unsaturated C5-dicarboxylic acid, has attracted much attention as a biobased building block chemical. It is produced industrially (>80 g L^?1) from glucose by fermentation with Aspergillus terreus. The titer is low compared with citric acid production (>200 g L^?1). This review summarizes the latest progress on enhancing the yield and productivity of IA production. IA biosynthesis involves the decarboxylation of the TCA cycle intermediate cis-aconitate through the action of cis-aconitate decarboxylase (CAD) enzyme encoded by the CadA gene in A. terreus. A number of recombinant microorganisms have been developed in an effort to overproduce it. IA is used as a monomer for production of superabsorbent polymer, resins, plastics, paints, and synthetic fibers. Its applications as a platform chemical are highlighted. It has a strong potential to replace petroleum-based methylacrylic acid in industry which will create a huge market for IA.     

Lovastatin Biosynthesis by Aspergillus terreus in a Chemically Defined Medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

Parameter optimization for thermostable lipase production and performance evaluation as prospective detergent additive

Abstract

Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R²?=?0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4?IU/g at an optimum temperature of 57.5?°C, pH 8.31, moisture 50% and 1.2?mg of bio-surfactant. Experimental production of 30.3?IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.     

Sequential optimization and large scale production of lipase using tri-substrate mixture from Aspergillus niger MTCC 872 by solid state fermentation

Tri-substrate mixture of Prosopis juliflora (PJ), red gram husk (RGH) and cotton seed cake (CSC) has been studied for the production of lipase (E.C. 3.1.1.3) using Aspergillus niger MTCC 872 in solid state fermentation. Simplex centroid mixture design (SCMD) was implemented to optimize the tri-substrate mixture composition consisting of PJ, RGH and CSC. Mixture taken in the ratio of 6.66:1.66:1.66 for PJ:RGH:CSC has shown highest lipase activity of 212.20 ± 6.36 U/gds at 30 °C, 7 pH and 70 % initial moisture content (v/w). Sequential optimization of physical parameters was done using the central composite face-centered design (CCFD). The optimum mixture composition has shown the highest lipase activity of 269.87 ± 8.09 U/gds at 35 °C, 7 pH and 70 % initial moisture content (v/w). ANOVA analysis for SCMD and CCFD confirms the model’s significance with R² values of 0.9989 and 0.968. A 1.27 fold increased lipase activity was obtained after physical parameters optimization. Large scale production using 1 kg substrate was carried out in tray bioreactor with different bed heights and the highest lipase activity of 208.79 ± 6.26 U/gds was obtained. This study signifies the enhancement of lipase production using substrate PJ for lipase production along with the other agricultural residues.     

Cloning, expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin A esters

Lipase is one of the most important industrial enzymes, which has been widely used in the preparation of food additives, cosmetics and pharmaceuticals industries. In order to obtain a large amount of lipase, the lipase gene from Candida antarctica ZJB09193 was cloned, and expressed in Pichia pastoris with the vector pPICZαA. Under the optimal conditions, the yield of recombinant lipase in the culture broth reached 3.0 g/L. After purification, the properties of recombinant lipase were studied: the optimum pH and temperature were pH 8.0 and 52°C, Ca(2+) activated the activity of lipase, and the apparent K(m) and V(max) values for p-nitrophenyl acetate were 0.34 mM and 7.36 μmol min(-1) mg(-1), respectively. Furthermore, the recombinant lipase was immobilized on pretreated textile for biosynthesis of vitamin A esters. In a system of n-hexane, 0.3 g immobilized recombinant lipase was used in the presence of 0.06 g vitamin A acetate and 0.55 mmol fatty acid (nine different fatty acids were tested). The yield of all vitamin A esters exceeded 78% in 7h at 30°C except using lactic acid and hexanoic acid as substrates. After optimization, the yield of vitamin A palmitate reached 87%. This study has the potential to be developed into industrial application.     

Enhanced production of (+)-terrein by Aspergillus terreus strain PF26 with epigenetic modifier suberoylanilide hydroxamic acid

New strategies for improving the fermentation yield of (+)-terrein which is a fungal metabolite with multiple bioactivities are very urgent. In this study, the effect of suberoylanilide hydroxamic acid, one kind of epigenetic modifier, on the biosynthesis of (+)-terrein by Aspergillus terreus strain PF26 isolated from the marine sponge Phakellia fusca was investigated. It was found that suberoylanilide hydroxamic acid exhibited a positive impact on (+)-terrein production, resulting from promoting the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein. Through optimization of feeding concentration and time of suberoylanilide hydroxamic acid, 5.58 g/L (+)-terrein could be obtained in shake flask cultivation, 29.5% higher than the control. Correspondingly, the fermentation of A. terreus strain PF26 in 7.5-L stirred bioreactor with feeding suberoylanilide hydroxamic acid (900 μM, day 4) yielded 9.07 g/L (+)-terrein, 77.1% higher than the control. These results showed that the epigenetic modifier-suberoylanilide hydroxamic acid could be utilized to enhance the production of (+)-terrein, which laid the foundation of massive production of (+)-terrein by fermentation.     

Pongamia pinnata seed cake: a promising and inexpensive substrate for production of protease and lipase from Bacillus pumilus SG2 on solid-state fermentation

The production of a protease and a lipase from Bacillus pumilus SG2 on solid-state fermentation using Pongamia pinnata seed cake as substrate was studied. The seed cake was proved to be a promising substrate for the bacterial growth and the enzyme production. The initial pH, incubation time and moisture content were optimized to achieve maximal enzyme production. Maximum protease production was observed at 72 h and that of the lipase at 96 h of incubation. The production of protease (9840 U/g DM) and lipase (1974 U/g DM) were maximum at pH 7.0 and at 60% moisture content. Triton X-100 (1%) was proved to be an effective extractant for the enzymes and their optimal activity was observed at alkaline pH and at 60 C. The molecular mass of the protease and lipase was 24 and 40 kDa, respectively. Both the enzymes were found to be stable detergent additives. The study demonstrated that inexpensive and easily available Pongamia seed cake could be used for production of industrially important enzymes, such as protease and lipase.     

Statistical optimization for lipase production from solid waste of vegetable oil industry

The production of biofuel using thermostable bacterial lipase from hot spring bacteria out of low-cost agricultural residue olive oil cake is reported in the present paper. Using a lipase enzyme from Bacillus licheniformis, a 66.5% yield of methyl esters was obtained. Optimum parameters were determined, with maximum production of lipase at a pH of 8.2, temperature 50.8°C, moisture content of 55.7%, and biosurfactant content of 1.693?mg. The contour plots and 3D surface responses depict the significant interaction of pH and moisture content with biosurfactant during lipase production. Chromatographic analysis of the lipase transesterification product was methyl esters, from kitchen waste oil under optimized conditions, generated methyl palmitate, methyl stearate, methyl oleate, and methyl linoleate.     

Uptake and accumulation of B-group vitamers in Saccharomyces cerevisiae in ethanol-stat fed-batch culture

The uptake and accumulation of the B-group vitamins thiamine, riboflavin, nicotinamide, pantothenic acid and pyridoxine in Saccharomyces cerevisiae was studied by gradually increasing the specific dosage of vitamins in an ethanol-stat fed-batch culture. Thiamine, nicotinamide, pantothenic acid, and pyridoxine were almost completely taken up at low vitamin dosages. Thiamine was determined to be the major accumulating form of vitamin B₁ while most of the assimilated nicotinamide and pantothenic acid accumulated in cofactor forms. Despite the obvious uptake of pyridoxine, accumulation of B₆ vitamers was not observed. In contrast with the other vitamins studied, riboflavin began accumulating in the culture medium immediately after vitamin addition was initiated. By the end of the experiment, the apparent uptake of all vitamins exceeded their accumulation in the cells. Variations in the growth rate of yeast at different vitamin dosages demonstrate the importance of balancing the vitamins in the media during cultivation.     

Lipase production and Penicillium simplicissimum morphology in solid-state and submerged fermentations

A comparative study of Penicillium simplicissimum morphology and lipase production was performed using solid-state (SSF) and submerged (SmF) fermentation. SSF was carried out on babassu cake as culture medium and SmF on a semi-synthetic medium and a medium based on suspended babassu cake grains. Yield of product on biomass, specific activity and conidia production were 3.3-, 1.3- and 2-fold higher in SSF. In SmF, the type of fungus growth differed according to the medium. Using the semi-synthetic medium, the fungus formed densely interwoven mycelial masses without conidia production, whereas using the babassu-based medium the fungus formed free mycelia and adhered to the surfaces of the grains, producing conidia. The results show that babassu cake induces conidiation in SmF. In SSF, the fungus not only grew on the surface of the grains, producing conidia abundantly, but also effectively colonized and penetrated the babassu particles. The high conidia production and lipase productivity in SSF may be related to the low availability of nutrients or to other stimuli associated with this type of fermentation. Thus, the high production of the thermostable P. simplicissimum lipase, using a non-supplemented, low-cost agro-industrial residue as the culture medium, demonstrates the biotechnological potential of SSF for the production of industrial enzymes.     

Reduced by-product formation and modified oxygen availability improve itaconic acid production in Aspergillus niger

Aspergillus niger has an extraordinary potential to produce organic acids as proven by its application in industrial citric acid production. Previously, it was shown that expression of the cis-aconitate decarboxylase gene (cadA) from Aspergillus terreus converted A. niger into an itaconic acid producer (Li et al., Fungal Genet Bio 48: 602–611, 2011). After some initial steps in production optimization in the previous research (Li et al., BMC biotechnol 12: 57, 2012), this research aims at modifying host strains and fermentation conditions to further improve itaconic acid production. Expression of two previously identified A. terreus genes encoding putative organic acid transporters (mttA, mfsA) increased itaconic acid production in an A. niger cis-aconitate decarboxylase expressing strain. Surprisingly, the production did not increase further when both transporters were expressed together. Meanwhile, oxalic acid was accumulated as a by-product in the culture of mfsA transformants. In order to further increase itaconic acid production and eliminate by-product formation, the non-acidifying strain D15#26 and the oxaloacetate acetylhydrolase (oahA) deletion strain AB 1.13 ?oahA #76 have been analyzed for itaconic acid production. Whereas cadA expression in AB 1.13 ?oahA #76 resulted in higher itaconic acid production than strain CAD 10.1, this was not the case in strain D15#26. As expected, oxalic acid production was eliminated in both strains. In a further attempt to increase itaconic acid levels, an improved basal citric acid-producing strain, N201, was used for cadA expression. A selected transformant (N201CAD) produced more itaconic acid than strain CAD 10.1, derived from A. niger strain AB1.13. Subsequently, we have focused on the influence of dissolved oxygen (D.O.) on itaconic acid production. Interestingly, reduced D.O. levels (10–25 %) increased itaconic acid production using strain N201 CAD. Similar results were obtained in strain AB 1.13 CAD + HBD2.5 (HBD 2.5) which overexpressed a fungal hemoglobin domain. Our results showed that overexpression of the hemoglobin domain increased itaconic acid production in A. niger at lower D.O. levels. Evidently, the lower levels of D.O. have a positive influence on itaconic acid production in A. niger strains.     

Supplementation of the cultivation media with B-group vitamins enhances lovastatin biosynthesis by Aspergillus terreus

The impact of the supplementation of cultivation media with B-group vitamins on the biosynthesis of lovastatin (mevinolinic acid) by Aspergillus terreus ATCC20542 was investigated. A hypothesis was formulated that as the biosynthesis of lovastatin requires a high throughput of coenzymes in the cells, the application of its precursors in the form of B-group vitamins might positively influence the process. In a nitrogen-deficient medium the B-group vitamins, both single, especially nicotinamide, pyridoxine and calcium D-pantothenate, and a mixture of thiamine, riboflavin, pyridoxine, calcium d-pantothenate and nicotinamide increased the efficiency of lovastatin biosynthesis. The vitamin supplementation also increased both volumetric and specific production rates of mevinolinic acid, especially before 80 h of the process, when no lactose limitation had been observed yet.     

Vitamin B6 Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B₆) as a cofactor. This vitamin can be synthesized by tsetse''s obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B₆ for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse''s reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B₆ metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B₆ in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B₆ production.     

Isolation of bioactive peptides from tryptone that modulate lipase production in Yarrowia lipolytica

In this work the effect of several organic nitrogen sources on lipase production in Yarrowia lipolytica LgX64.81 overproducing mutant was studied. Among them, tryptone and peptone showed the most prominent stimulatory effect. Interestingly, only tryptic and peptic casein digest were found to highly induce lipase biosynthesis while lipase production was very limited in the presence of casein digest from papain and pronase-catalysed hydrolysis and absent in case of chymotryptic digest. It was also demonstrated that the stimulatory peptides should be present in the culture medium at specific proportions and molecular size to match the physiological requirement of Yarrowia lipolytica strain for lipase biosynthesis.     

Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate

Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO(3) for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production.     

Research on the ability of propionic acid and vitamin B12 biosynthesis by <Emphasis Type="Italic">Propionibacterium freudenreichii</Emphasis> strain T82

The purpose of this study was to determine the potential for biosynthesis of propionic acid and vitamin B12 by Propionibacterium freudenreichii T82 in a medium containing various sources of carbon (glucose, fructose, and saccharose). These sugars are present in apple pomaces, which are the waste from the production of apple juice. Using statistical analysis design of experiments (DoE), the results allowed us to determine which sugars (carbon sources) exert the most beneficial influence on the biosynthesis of propionic acid and cobalamin. The highest production of propionic acid by the tested bacterial strain was obtained in a medium in which glucose accounted for at least 50% of the available carbon sources. Depending on the culture medium, the concentration of this metabolite ranged from 23 to 40 g/L. P. freudenreichii T82 produced the smallest amount of acid in medium in which the dominant nutrient source was saccharose. The results obtained indicated an inverse relationship between the amount of acid produced by the bacteria and vitamin B12 biosynthesis. Because of the high efficiency of propionic acid biosynthesis by P. freudenreichii T82, the prospect of using this strain to obtain propionate with the simultaneous disposal of waste materials (such as apple pomaces) which contain glucose and/or fructose is very promising.