Production Of Caffeic Acid Research Articles

Metabolic engineering strategies for caffeic acid production in Escherichia coli

Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is an aromatic compound obtained by the phenylpropanoid pathway. This natural product has antioxidant, antitumor, antiviral, and anti-inflammatory activities. It is also a precursor of CA phenethyl ester (CAPE), a compound with potential as an antidiabetic and liver-protective agent. CA can be found at low concentrations in plant tissues, and hence, its purification is difficult and expensive. Knowledge regarding the pathways, enzymes, and genes involved in CA biosynthesis has paved the way for enabling the design and construction of microbial strains with the capacity of synthesizing this metabolite. In this review, metabolic engineering strategies for the generation of Escherichia coli strains for the biotechnological production of CA are presented and discussed.

Active compounds and biological activity of in vitro cultures of some Echinacea purpurea varieties

BackgroundPlant cell cultures have developed rapidly in recent years for the synthesis of selected natural products. This study was conducted to evaluate the effect of yeast extract, jasmonic acid, phenylalanine, and tyrosine on total caffeic acid and polysaccharide production in callus cultures of two Echinacea varieties, namely Double Decker and Rubinstern. The biological activities (antioxidant, antimicrobial and cytotoxic activities) of different Echinacea extracts were also evaluated.ResultsData revealed the effect of yeast extract on calli of the two varieties. Double Decker calli recorded maximum caffeic acid derivatives and total hydrolysable sugars after 30 days of cultivation using 1.5 g/l and 1.0 g/l yeast extract respectively whereas, in Rubinstern calli, the maximum caffeic acid derivatives and total hydrolysable sugars were recorded at 1.0 g/l after 30 days. Using 50 mM jasmonic acid, Double Decker calli recorded maximum values of caffeic acid derivatives and total hydrolysable sugars after 15 days. In Rubinstern calli, caffeic acid derivatives and total hydrolysable sugar recorded maximum values after 30 days at 200 mM jasmonic acid and after 15 days at 50 mM jasmonic acid, respectively. In Double Decker variety, caffeic acid derivatives and total hydrolysable sugars recorded maximum values at 100 mg/l phenylalanine. Rubinstern calli recorded maximum value of caffeic acid derivatives at 100 mg/l phenylalanine and total hydrolysable sugars at 50 mg/l phenylalanine. As for tyrosine, maximum values of caffeic acid derivatives and total hydrolysable sugars recorded at 150 mg/l with Double Decker calli. Rubinstern calli recorded maximum value of caffeic acid derivatives and total hydrolysable sugars at 150 and 50 mg/l tyrosine, respectively. The biological activities of the different Echinacea extracts showed that maximum antioxidant activity (89.2%) was recorded with Rubinstern calli. Also, the maximum value of cell death (78.2%) was observed with the extract of Rubinstern calli. For antibacterial activity, most extracts showed inhibitory effect against Bacillus subtilis and Staphylococcus aureus growth.ConclusionBoth elicitors (yeast extract and jasmonic acid) and precursors (phenylalanine and tyrosine) have a clear effect on natural products of the two Echinacea varieties. The investigated Echinacea extracts (in vitro and in vivo plants and calli of the two varieties) showed moderate activity against tested microbial strains.

Effect of salicylic acid and methyl jasmonate on the production of rosmarinic acid and caffeic acid in callus culture of Salvia lerrifolia Benth

در پژوهش حاضر، کالوس‌زایی گیاه دارویی نوروزک (Salvia leriifolia Benth.) با جدا‌کشت ساقه و برگ در ترکیب اکسین‌های 1 - نفتالن استیک اسید (NAA) و2, 4-D با سیتوکینین Kin و سپس اثر الیسیتورهای سالیسیلیک اسید و متیل جاسمونات (50، 100، 150 میکرومولار) در کالوس بر محتوای برخی متابولیت‌های ثانویه بررسی شدند. 1 – نفتالن استیک اسید با Kin در جداکشت ساقه و برگ کالوس‌زایی نکرد؛ درحالی‌که با جدا‌کشت برگ در غلظت 1 میلی‌گرم بر لیتر Kin با 2 میلی‌گرم بر لیتر 2, 4-D مناسب‌ترین کالوس تولید شد. بیشترین میزان وزن تر و خشک، محتوای فنل کل و فلاونوئید در کالوس‌های تیمار‌شده با 100 میکرو‌مولار متیل‌جاسمونات مشاهده شدند؛ باوجوداین با افزایش غلظت سالیسیلیک اسید، وزن تر و خشک، محتوای فنل کل، فلاونوئید، رزمارینیک اسید روند رو‌به‌افزایش داشتند و بیشترین مقدار رزمازینیک اسید و کافئیک اسید به‌ترتیب در کالوس‌های تیمارشده با سالیسیلیک اسید 100 و 150 میکرومولار مشاهده شدند. نتایج نشان دادند بهینه‌کردن غلظت الیسیتورها، بر تولید متابولیت‌های ثانویة مدنظر در نوروزک در شرایط درون‌شیشه‌ای تأثیر می‌گذارد.

Characterization and application of fungal chlorogenate hydrolase to enzymatic breaking down of chlorogenate from yerba mate

Abstract The aim of this work was to study the physicochemical and kinetic properties of chlorogenate hydrolase (CHase) (EC 3.1.1.42) of Aspergillus niger AKU 3302 by induction in the presence of yerba mate extract. The data obtained could be applicable directly to mass processing for a plant scale production of quinic acid (QA) and caffeic acid (CA) with koji CHase. It is fundamentally significant to accumulate catalytic properties of CHase as much as possible with crude CHase, when koji CHase is directed to practical industrial processing of breakdown of chlorogenate (CGA). CHase exhibited a high thermal stability without appreciable loss after standing for 6 h at 50 °C and was stable in wide pH ranges from 2.0 to 9.0. The optimum pH and temperature of CHase activity were found at 6.5–7.0 and 45 °C, respectively. The Vmax and Km values for CGA hydrolysis were 1.996 unit mg−1 and 0.72 mM, respectively. The activation energy of CHase was estimated to be 26 kJ mol−1. Thus, CHase induced with CGA revealed successful for the production of CA and QA, fine chemicals of high interest and commercial values, from natural plant resources rich in CGA such as yerba mate. Several advantageous merits obtained with a crude CHase for the practical formation of QA and CA from yerba mate or yerba mate extract are discussed.

Hydroxycinnamic acids and curcumin production in engineered Escherichia coli using heat shock promoters

Hydroxycinnamic acids and curcumin are compounds with great therapeutic potential, including anticancer properties. In this study, p-coumaric acid, caffeic acid and curcumin were produced in Escherichia coli. Their production was induced by heat using the dnaK and ibpA heat shock promoters. The ribosome binding site (RBS) used was tested and further optimized for each gene to assure an efficient translation. p-Coumaric acid was successfully produced from tyrosine and caffeic acid was produced either from tyrosine or p-coumaric acid using tyrosine ammonia lyase (TAL) from Rhodotorula glutinis, 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris. The highest p-coumaric acid production obtained was 2.5mM; caffeic acid production reached 370μM. Regarding curcumin, 17μM was produced using 4-coumarate-CoA ligase (4CL1) from Arabidopsis thaliana, diketide-CoA synthase (DCS) and curcumin synthase 1 (CURS1) from Curcuma longa. Stronger RBSs and/or different induction conditions should be further evaluated to optimize those production levels. Herein it was demonstrated that the biosynthetic pathway of p-coumaric acid, caffeic acid and curcumin in E. coli can be triggered by using heat shock promoters, suggesting its potential for the development of new industrial bioprocesses or even new bacterial therapies.

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli.

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233mg/L) far exceeded that by SHF (37.9mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

Responses of Crepis japonica induced by supplemental blue light and UV-A radiation

Crepis japonica (L.) D.C. (Asteraceae), a weed with antioxidant, antiallergenic, antiviral and antitumor properties displays both medicinal properties and nutritional value. This study aims to assess the effects of a supplementation of blue light and UV-A radiation on the growth, leaf anatomical structure and phenolic profile of the aerial parts of Crepis japonica. Plants were grown under two light treatments: W (control - white light), W + B (white light supplemented with blue light) and W + UV-A (white light supplemented with UV-A radiation). We recorded the length, width, and weight of fresh and dry leaves, the thickness of the epidermis and mesophyll, and stomata density. The phenolic profiles of the aqueous extracts of the aerial parts were analyzed by HPLC-DAD. There was an increase in the leaf size, stomatal density, and phenolic production, and a thickening of the mesophyll and epidermis. UV-A radiation increased the phenolic production more than blue light. Blue light and UV-A radiation both improved the production of caffeic acid by about 6 and 3 times, respectively, in comparison to control. This compound was first reported as a constituent of the extract from the aerial parts together with caftaric acid. UV-A also promoted the production of chlorogenic acid (about 1.5 times in comparison to the control). We observed that the morphological and chemical parameters of C. japonica are modified in response to blue light and UV-A radiation, which can be used as tools in the cultivation of this species in order to improve its medicinal properties and nutritional value.

Rhizophagus irregularis as an elicitor of rosmarinic acid and antioxidant production by transformed roots of Ocimum basilicum in an in vitro co-culture system.

Arbuscular mycorrhiza is a symbiotic association formed between plant roots and soil borne fungi that alter and at times improve the production of secondary metabolites. Detailed information is available on mycorrhizal development and its influence on plants grown under various edapho-climatic conditions, however, very little is known about their influence on transformed roots that are rich reserves of secondary metabolites. This raises the question of how mycorrhizal colonization progresses in transformed roots grown in vitro and whether the mycorrhizal fungus presence influences the production of secondary metabolites. To fully understand mycorrhizal ontogenesis and its effect on root morphology, root biomass, total phenolics, rosmarinic acid, caffeic acid and antioxidant production under in vitro conditions, a co-culture was developed between three Agrobacterium rhizogenes-derived, elite-transformed root lines of Ocimum basilicum and Rhizophagus irregularis. We found that mycorrhizal ontogenesis in transformed roots was similar to mycorrhizal roots obtained from an in planta system. Mycorrhizal establishment was also found to be transformed root line-specific. Colonization of transformed roots increased the concentration of rosmarinic acid, caffeic acid and antioxidant production while no effect was observed on root morphological traits and biomass. Enhancement of total phenolics and rosmarinic acid in the three mycorrhizal transformed root lines was found to be transformed root line-specific and age dependent. We reveal the potential of R. irregularis as a biotic elicitor in vitro and propose its incorporation into commercial in vitro secondary metabolite production via transformed roots.

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62mM (472mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56mM (280mg/L) and 1mM (180mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.

Selected bioactives from callus cultures of olives (Olea europaea L. Var. Coratina) by LC-MS

Olive biophenols, OBPs, and biophenol-secoiridoids, OBPsecos, soluble bioactives from olives, and then in olive oil and table olives, were biosynthesized from Coratina olive callus cultures, under bio- and techno-mimetic experimental conditions. We detected major production of OBPs, hydroxytyrosol, tyrosol, caffeic acid and verbascoside, and of OBP-secos, oleuropein and ligstroside, together with minor amounts of oleuroside, and of demethyl and deglucosil bioactives, by high-performance liquid chromatography, HPLC, coupled with electrospray ionization mass spectrometry, ESI-MS. The olive callus system, in an experimental non-pathogenic environment, produced a nearly model balance of OBPs and OBPsecos in suitable amount comparable to drupes, leaves and seeds. The bioreactor scaling-up of more homogeneous and rapidly growing systems, processed by callus shaking to suspension cell cultures, should produce nutraceutical and cosmeceutical bioactives.

Functional expression of an Arabidopsis p450 enzyme, p-coumarate-3-hydroxylase, in the cyanobacterium Synechocystis PCC 6803 for the biosynthesis of caffeic acid

Caffeic acid, which exhibits strong anticancer activities, is a natural phenolic compound found in small amounts in plants. Production of caffeic acid by bacterial systems is technically challenging due to difficulties in functionally expressing p-coumarate-3-hydroxylase (C3H), a cytochrome P450 enzyme that converts p-coumaric acid into caffeic acid. Here, we report for the first time that the cyanobacterium Synechocystis PCC 6803 is able to produce caffeic acid from p-coumaric acid upon heterologous expression of C3H. The Arabidopsis thaliana ref8 gene, which encodes a C3H, was synthesized and codon-optimized for enhanced expression in Synechocystis. Expression of the synthetic ref8 gene was driven by a native psbA2 promoter and confirmed at the transcriptional and translational levels. This heterologous pathway enabled Synechocystis to produce caffeic acid at a concentration of ∼7.2 mg L−1 from p-coumaric acid under oxygenic photosynthetic growth conditions. This study demonstrates that cyanobacteria are well suited for the bioproduction of plant secondary metabolites that are difficult to produce in other bacterial systems.

Caffeic acid production enhancement by engineering a phenylalanine over‐producing Escherichia coli strain

Caffeic acid is a plant-specific phenylpropanoic acid with multiple health-improving effects reported, and its therapeutic derivatives have also been studied throughout the last decade. To meet its market need and achieve high-level production, microbial production of caffeic acid approaches have been developed in metabolically engineered Escherichia coli. In our previous work, we have established the first artificial pathway that realized de novo production of caffeic acid using E. coli endogenous 4-hydroxyphenylacetate 3-hydroxylase (4HP3H). In this work, we exploited the catalytic potential of 4HPA3H in the whole-cell bioconversion study and produced 3.82 g/L (461.12 mg/L/OD) caffeic acid from p-coumaric acid, a direct precursor. We further engineered a phenylalanine over-producer into a tyrosine over-producer and then introduced the artificial pathway. After adjusting the expression strategy and optimizing the inoculants timing, de novo production of caffeic acid reached 766.68 mg/L. Both results from the direct precursor and simple carbon sources represent the highest titers of caffeic acid from microbial production so far.

Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives

4-Hydroxyphenylacetate 3-hydroxylases (HPAHs) of the two-component flavin-dependent monooxygenase family are attractive enzymes that possess the catalytic potential to synthesize valuable ortho-diphenol compounds from simple monophenol compounds. In this study, we investigated the catalytic activity of HPAH from Pseudomonas aeruginosa strain PAO1 toward cinnamic acid derivatives. We prepared Escherichia coli cells expressing the hpaB gene encoding the monooxygenase component and the hpaC gene encoding the oxidoreductase component. E. coli cells expressing HpaBC exhibited no or very low oxidation activity toward cinnamic acid, o-coumaric acid, and m-coumaric acid, whereas they rapidly oxidized p-coumaric acid to caffeic acid. Interestingly, after p-coumaric acid was almost completely consumed, the resulting caffeic acid was further oxidized to 3,4,5-trihydroxycinnamic acid. In addition, HpaBC exhibited oxidation activity toward 3-(4-hydroxyphenyl)propanoic acid, ferulic acid, and coniferaldehyde to produce the corresponding ortho-diphenols. We also investigated a flask-scale production of caffeic acid from p-coumaric acid as the model reaction for HpaBC-catalyzed syntheses of hydroxycinnamic acids. Since the initial concentrations of the substrate p-coumaric acid higher than 40 mM markedly inhibited its HpaBC-catalyzed oxidation, the reaction was carried out by repeatedly adding 20 mM of this substrate to the reaction mixture. Furthermore, by using the HpaBC whole-cell catalyst in the presence of glycerol, our experimental setup achieved the high-yield production of caffeic acid, i.e., 56.6 mM (10.2 g/L) within 24 h. These catalytic activities of HpaBC will provide an easy and environment-friendly synthetic approach to hydroxycinnamic acids.

Engineering E. coli for caffeic acid biosynthesis from renewable sugars

Caffeic acid is a valuable aromatic compound that possesses many important pharmacological activities. In structure, caffeic acid belongs to the hydroxycinnamic acid family and can be biosynthesized from the aromatic amino acid tyrosine. In the present paper, the caffeic acid biosynthesis pathway was reconstituted in engineered Escherichia coli to produce caffeic acid from simple biomass sugar glucose and xylose. Different engineering approaches were utilized to optimize the production. Specifically, two parallel biosynthesis routes leading from tyrosine to caffeic acid were studied. The copy number of the intermediate biosynthesis genes was varied to find appropriate gene doses for caffeic acid biosynthesis. Three different media, including a MOPS medium, a synthetic medium, and a rich medium, were also examined to improve the production. The highest specific caffeic acid production achieved was 38 mg/L/OD. Lastly, cultivation of engineered E. coli in a bioreactor resulted in a production of 106 mg/L caffeic acid after 4 days.

Production of low chlorogenic and caffeic acid containing sunflower meal protein isolate and its use in functional wheat bread making.

Sunflower meal protein isolate (SMPI) is a promising food additive in different matrices. However, the uses of SMPI are limited because of the presence of antinutritional compounds like polyphenolic substances. Chlorogenic and caffeic acids are the dominants polyphenolics in the SMPI. These substances cause significant changes of the colour of the meal, proteins and food matrices during their extraction and use as food additives. Moreover, these substances lower the nutritional value of the end product due to their interaction with some amino acids such as lysine and methionine. Thus, the removal of these substances is important to enable the use of the SMPI and meal in general in a greater extent in food applications and replacing more expensive protein sources such as soy proteins. The aim of this work was to study the production of functional bread by supplementing wheat flour with sunflower meal protein isolate (SMPI). SMPI with low content of chlorogenic and caffeic acid was usefully produced following alkaline extraction and purification with succinic acid. Purified SMPI showed well balanced amino acid profile and was characterized by high water and fat absorption capacities. It was incorporated to dough formula at 8-12% of the total wheat flour. The results showed that production of bread supplemented with SMPI was technologically feasible. The supplemented bread had high mass volume and nutritional quality compared to the control bread. The optimal SMPI to incorporate into dough formula without significant alteration of the final bread colour was established at 10%. This study will be helpful to find economic ways to enhance the nutritional quality of wheat bread and to improve the profitability of sunflower meal residue.

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.

Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

BackgroundCaffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE) have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources.ResultsWe first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H), is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs) from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc) possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L) in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation.ConclusionWe have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from caffeic acid. In addition, we have identified that TAL is the rate-limiting enzyme in this pathway. Thus, exploration for more active TALs via bio-prospecting and protein engineering approaches is necessary for further improvement of caffeic acid production.

Application of response surface methodology optimization for the production of caffeic acid from tobacco waste

Caffeic acid (CA) was widely applied in food and pharmaceutical industries as an important natural antioxidant. In the present study, the main caffeoylquinic acids (CQAs) in tobacco waste were analyzed by HPLC-PAD-ESI-MS/MS, and used as raw material for producing CA which preparation process was optimized by response surface methodology (RSM). The research results indicated the main ingredients of CQAs in tobacco waste were identified as three isomers containing chlorogenic acid (5-caffecylquinic acid, 5-CQA), cryptochlorogenic acid (4-caffecylquinic acid, 4-CQA) and neochlorogenic acid (3-caffecylquinic acid, 3-CQA), respectively. The production of CA produced by alkaline hydrolysis of the CQAs extract from tobacco waste was effectively enhanced by RSM. NaOH was selected as catalyst through screening of four different alkalis. Three hydrolysis reaction parameters for production of CA were optimized as follows by using central composite rotatable design (CCRD) of RSM: pH value 13.5, reaction temperature 85oC and reaction time 10 min. The adequately high R2 value 0.9275 and F score 7.105 indicated the statistical significance of the model. The conversion rate of CA after optimization was 43.46% while the predicted value was 44.92%. In conclusion, hydrolysis conditions optimization to enhance CA production from tobacco waste can be easily and effectively done by RSM; the developed production process of CA indicated CQAs in tobacco waste are potential raw material for producing CA.

Enzymatic Production of Caffeic Acid by Koji from Plant Resources Containing Caffeoylquinic Acid Derivatives

The effect of a koji (Aspergillus awamori mut.) extract on the caffeoylquinic acid derivatives purified from sweetpotato (Ipomoea batatas L.) leaves was examined to develop the mass production of caffeic acid. A koji extract hydrolyzed the caffeoylquinic acid derivatives, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid, to caffeic acid. Furthermore, the koji extract also converted the major polyphenolic components from sweetpotato, burdock (Arctium lappa L.), and mugwort (Artemisia indica var. maximowiczii) leaves to caffeic acid. These results suggest that the production of caffeic acid from plant resources containing caffeoylquinic acid derivatives is possible.

Purification and characterization of a chlorogenic acid hydrolase from Aspergillus niger catalysing the hydrolysis of chlorogenic acid

Among 15 Aspergillus strains, Aspergillus niger BRFM 131 was selected for its high chlorogenic acid hydrolase activity. The enzyme was purified and characterized with respect to its physico-chemical and kinetic properties. Four chromatographic steps were necessary to purify the protein to homogeneity with a recovery of 2%. K m of the chlorogenic acid hydrolase was estimated to be 10 μM against chlorogenic acid as substrate. Under native conditions, the protein presented a molecular mass of 170 kDa, and SDS-PAGE analysis suggested the presence of two identical 80 kDa subunits. Isoelectric point was 6.0; pH optimum for activity was determined to be 6.0 and temperature optima to be 55 °C. The N-terminal sequence did not present any homology with other cinnamoyl ester hydrolases previously described suggesting the purification of a new protein. The chlorogenic acid hydrolase was used successfully for the production of caffeic acid, which possesses strong antioxidant properties, from natural substrates specially rich in chlorogenic acid like apple marc and coffee pulp.