Spectrophotometric and Fluorimetric High-Throughput Assays for Phenolic Acid Decarboxylase.

Biocatalytic decarboxylation of hydroxycinnamic acids yields phenolic styrenes, which are important precursors for antioxidants, epoxy coatings, adhesives and other polymeric materials. Bacillus subtilis decarboxylase (BsPAD) is a cofactor-independent enzyme that catalyzes the cleavage of carbon dioxide from p-coumaric-, caffeic-, and ferulic acid with high catalytic efficiency. Real-time spectroscopic assays for decarboxylase reactions remove the necessity of extensive sample workup, which is required for HPLC, mass spectrometry, gas chromatography, or NMR methods. This work presents two robust and sensitive assays based on photometry and fluorimetry that allow decarboxylation reactions to be followed with high sensitivity while avoiding product extraction and long analysis times. Optimized assay procedures were used to measure BsPAD activity in cell lysates and to determine the kinetic constants (KM and Vmax ) of the purified enzyme for p-coumaric-, caffeic- and ferulic acid. Substrate inhibition was shown for caffeic acid.

Improving the catalytic characteristics of phenolic acid decarboxylase from Bacillus amyloliquefaciens by the engineering of N-terminus and C-terminus.

BACKGROUND: 4-vinylphenols produced by phenolic acid degradation catalyzed by phenolic acid decarboxylase can be used in food additives as well as flavor and fragrance industry. Improving the catalytic characters of phenolic acid decarboxylase is of great significance to enhance its practical application. RESULTS: A phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; however, P-N had reduced affinity toward p-coumaric acid. The extension of the C-terminus increased its acid resistance, whereas the extension of the N-terminus contributed to the alkali resistance and heat resistance. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. However, the affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min. CONCLUSIONS: The extension of the N-terminus may be more conducive to the combination of the binding cavity with the substrate in an alkaline environment and may make its structure more stable.

Genetic Determinants of Hydroxycinnamic Acid Metabolism in Heterofermentative Lactobacilli.

Phenolic acids are among the most abundant phenolic compounds in edible parts of plants. Lactic acid bacteria (LAB) metabolize phenolic acids, but the enzyme responsible for reducing hydroxycinnamic acids to phenylpropionic acids (HcrB) was only recently characterized in Lactobacillus plantarum In this study, heterofermentative LAB species were screened for their hydroxycinnamic acid metabolism. Data on strain-specific metabolism in combination with comparative genomic analyses identified homologs of HcrB as putative phenolic acid reductases. Par1 and HcrF both encode putative multidomain proteins with 25% and 63% amino acid identity to HcrB, respectively. Of these genes, par1 in L. rossiae and hcrF in L. fermentum were overexpressed in response to hydroxycinnamic acids. The deletion of par1 in L. rossiae led to the loss of phenolic acid metabolism. The strain-specific metabolism of phenolic acids was congruent with the genotype of lactobacilli; however, phenolic acid reductases were not identified in strains of Weissella cibaria that reduced hydroxycinnamic acids to phenylpropionic acids. Phylogenetic analysis of major genes involved in hydroxycinnamic acid metabolism in strains of the genus Lactobacillus revealed that Par1 was found to be the most widely distributed phenolic acid reductase, while HcrB was the least abundant, present in less than 9% of Lactobacillus spp. In conclusion, this study increased the knowledge on the genetic determinants of hydroxycinnamic acid metabolism, explaining the species- and strain-specific metabolic variations in lactobacilli and providing evidence of additional enzymes involved in hydroxycinnamic acid metabolism of lactobacilli.IMPORTANCE The metabolism of secondary plant metabolites, including phenolic compounds, by food-fermenting lactobacilli is a significant contributor to the safety, quality, and nutritional quality of fermented foods. The enzymes mediating hydrolysis, reduction, and decarboxylation of phenolic acid esters and phenolic acids in lactobacilli, however, are not fully characterized. The genomic analyses presented here provide evidence for three novel putative phenolic acid reductases. Matching comparative genomic analyses with phenotypic analysis and quantification of gene expression indicates that two of the three putative phenolic acid reductases, Par1 and HcrF, are involved in reduction of hydroxycinnamic acids to phenylpropionic acids; however, the activity of Par2 may be unrelated to phenolic acids and recognizes other secondary plant metabolites. These findings expand our knowledge on the metabolic potential of lactobacilli and facilitate future studies on activity and substrate specificity of enzymes involved in metabolism of phenolic compounds.

Metabolism of phenolic acids in whole wheat and rye malt sourdoughs.

This work aimed to study the phenolic acid metabolism of sourdough lactic acid bacteria (LAB) in laboratory media, and in sourdough fermentation with single cultures and in co-fermentations. Lactobacilli were selected from isolates obtained from 35 sourdough samples. Isolates (114 strains) were screened for phenolic acid decarboxylase gene pdc and EPS production. Ferulic acid metabolism of the 18 pdc positive strains was evaluated in mMRS; all pcd positive strains converted ferulic acid by decarboxylation and/or reduction. Single whole wheat and rye malt dough fermentation fermented with lactobacilli or yeasts were characterized with respect to free, conjugated, or bound phenolic acids. Concentrations of free, conjugated, or bound phenolic acids were not altered substantially in chemically acidified sourdoughs, or in yeast fermented doughs. L. plantarum metabolized free ferulic acid in wheat and rye malt sourdoughs; L. hammesii DSM 16381 metabolized syringic and vanillic acids and reduced levels of bound ferulic acid in wheat sourdoughs. Co-fermentation of L. hammesii and L. plantarum achieved release of bound ferulic acid and conversion of the resultant free ferulic acid to dihydroferulic acid and volatile metabolites. Phenolic acid metabolism in sourdoughs was enhanced by co-fermentation with strains exhibiting complementary metabolic activities. Results may enable improvement of bread quality by targeted conversion of phenolic acids during sourdough fermentation.

Structural and functional analysis of BF2549, a PadR-like transcription factor from Bacteroides fragilis.

A phenolic acid decarboxylase (padC) regulator, PadR and its homologs proteins belong to the PadR family. Despite the growing numbers of the PadR family members and their various roles in bacteria, such as detoxifications, drug transports and circadian rhythms, biochemical and biophysical studies of the PadR family are very limited. Thus, a ligand-induced regulatory mechanism of the PadR family transcription factors remains to be elucidated. Here, we report a crystal structure of a Bacteroides fragilis PadR-like protein, BF2549 and revealed its interaction with putative operator DNA and ligand molecules. Comparative structural and primary sequence analyses provide a PadR-specific motif that is conserved in the PadR family but deviated from the MarR family. Furthermore, putative ligand binding sites are observed in the BF2549 structure. Finally, a homology-based structure model of BF2549 and 29-mer dsDNA propose regulatory mechanisms of the PadR family in transcriptional derepression.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

Cloning and functional characterization of a phenolic acid decarboxylase from the liverwort Conocephalum japonicum.

Some commercially important vinyl derivatives are produced by the decarboxylation of phenolic acids. Enzymatically, this process can be achieved by phenolic acid decarboxylases (PADs), which are able to act on phenolic acid substrates such as ferulic and p-coumaric acid. Although many microbial PADs have been characterized, little is known regarding their plant homologs. Transcriptome sequencing in the liverworts has identified seven putative PADs, which share a measure of sequence identity with microbial PADs, but are typically much longer proteins. Here, a PAD-encoding gene was isolated from the liverwort species Conocephalum japonicum. The 1197 nt CjPAD cDNA sequence was predicted to be translated into a 398 residue protein. When the gene was heterologously expressed in Escherichia coli, its product exhibited a high level of PAD activity when provided with either p-coumaric or ferulic acid as substrate, along with the conversion of caffeic acid and sinapic acid to their corresponding decarboxylated products. Both N- and C-terminal truncation derivatives were non-functional. The transient expression in tobacco of a GFP/CjPAD fusion gene demonstrated that the CjPAD protein is expressed in the cytoplasm. It is first time a PAD was characterized from plants and the present investigation provided a candidate gene for catalyzing the formation of volatile phenols.

A New Phenolic Acid Decarboxylase from the Brown-Rot Fungus Neolentinus lepideus Natively Decarboxylates Biosourced Sinapic Acid into Canolol, a Bioactive Phenolic Compound.

Rapeseed meal (RSM) is a cheap, abundant and renewable feedstock, whose biorefinery is a current challenge for the sustainability of the oilseed sector. RSM is rich in sinapic acid (SA), a p-hydroxycinnamic acid that can be decarboxylated into canolol (2,6-dimethoxy-4-vinylphenol), a valuable bioactive compound. Microbial phenolic acid decarboxylases (PADs), mainly described for the non-oxidative decarboxylation of ferulic and p-coumaric acids, remain very poorly documented to date, for SA decarboxylation. The species Neolentinus lepideus has previously been shown to biotransform SA into canolol in vivo, but the enzyme responsible for bioconversion of the acid has never been characterized. In this study, we purified and characterized a new PAD from the canolol-overproducing strain N. lepideus BRFM15. Proteomic analysis highlighted a sole PAD-type protein sequence in the intracellular proteome of the strain. The native enzyme (NlePAD) displayed an unusual outstanding activity for decarboxylating SA (Vmax of 600 U.mg-1, kcat of 6.3 s-1 and kcat/KM of 1.6 s-1.mM-1). We showed that NlePAD (a homodimer of 2 × 22 kDa) is fully active in a pH range of 5.5-7.5 and a temperature range of 30-55 °C, with optima of pH 6-6.5 and 37-45 °C, and is highly stable at 4 °C and pH 6-8. Relative ratios of specific activities on ferulic, sinapic, p-coumaric and caffeic acids, respectively, were 100:24.9:13.4:3.9. The enzyme demonstrated in vitro effectiveness as a biocatalyst for the synthesis of canolol in aqueous medium from commercial SA, with a molar yield of 92%. Then, we developed processes to biotransform naturally-occurring SA from RSM into canolol by combining the complementary potentialities of an Aspergillus niger feruloyl esterase type-A, which is able to release free SA from the raw meal by hydrolyzing its conjugated forms, and NlePAD, in aqueous medium and mild conditions. NlePAD decarboxylation of biobased SA led to an overall yield of 1.6-3.8 mg canolol per gram of initial meal. Besides being the first characterization of a fungal PAD able to decarboxylate SA, this report shows that NlePAD is very promising as new biotechnological tool to generate biobased vinylphenols of industrial interest (especially canolol) as valuable platform chemicals for health, nutrition, cosmetics and green chemistry.

Challenges and advances in biotechnological approaches for the synthesis of canolol and other vinylphenols from biobased p-hydroxycinnamic acids: a review.

p-Hydroxycinnamic acids, such as sinapic, ferulic, p-coumaric and caffeic acids, are among the most abundant phenolic compounds found in plant biomass and agro-industrial by-products (e.g. cereal brans, sugar-beet and coffee pulps, oilseed meals). These p-hydroxycinnamic acids, and their resulting decarboxylation products named vinylphenols (canolol, 4-vinylguaiacol, 4-vinylphenol, 4-vinylcatechol), are bioactive molecules with many properties including antioxidant, anti-inflammatory and antimicrobial activities, and potential applications in food, cosmetic or pharmaceutical industries. They were also shown to be suitable precursors of new sustainable polymers and biobased substitutes for fine chemicals such as bisphenol A diglycidyl ethers. Non-oxidative microbial decarboxylation of p-hydroxycinnamic acids into vinylphenols involves cofactor-free and metal-independent phenolic acid decarboxylases (EC 4.1.1 carboxyl lyase family). Historically purified from bacteria (Bacillus, Lactobacillus, Pseudomonas, Enterobacter genera) and some yeasts (e.g. Brettanomyces or Candida), these enzymes were described for the decarboxylation of ferulic and p-coumaric acids into 4-vinylguaiacol and 4-vinylphenol, respectively. The catalytic mechanism comprised a first step involving p-hydroxycinnamic acid conversion into a semi-quinone that then decarboxylated spontaneously into the corresponding vinyl compound, in a second step. Bioconversion processes for synthesizing 4-vinylguaiacol and 4-vinylphenol by microbial decarboxylation of ferulic and p-coumaric acids historically attracted the most research using bacterial recombinant phenolic acid decarboxylases (especially Bacillus enzymes) and the processes developed to date included mono- or biphasic systems, and the use of free- or immobilized cells. More recently, filamentous fungi of the Neolentinus lepideus species were shown to natively produce a more versatile phenolic acid decarboxylase with high activity on sinapic acid in addition to the others p-hydroxycinnamic acids, opening the way to the production of canolol by biotechnological processes applied to rapeseed meal. Few studies have described the further microbial/enzymatic bioconversion of these vinylphenols into valuable compounds: (i) synthesis of flavours such as vanillin, 4-ethylguaiacol and 4-ethylphenol from 4-vinylguaiacol and 4-vinylphenol, (ii) laccase-mediated polymer synthesis from canolol, 4-vinylguaiacol and 4-vinylphenol.

Bioproduction of 4-Vinylphenol and 4-Vinylguaiacol ß-Primeverosides Using Transformed Bamboo Cells Expressing Bacterial Phenolic Acid Decarboxylase.

Phenolic acid decarboxylase (PAD) catalyzes the decarboxylation of hydroxycinnamic acids to produce hydroxystyrenes, which serve as starting materials for the production of polymers. Bamboo (Phyllostachys nigra; Pn) cells, a suitable host for producing phenylpropanoid-derived compounds, were transformed to express PAD of Bacillus amyloliquefaciens (BaPAD). BaPAD-transformed cells accumulated several metabolites that were not detected in wild-type Pn cells or BaPAD-negative transformant. Two major metabolites were isolated from BaPAD-transformed cells, and elucidation of their chemical structures confirmed these as 4-vinylphenol ß-primeveroside (4-VPP) and 4-vinylguaiacol ß-primeveroside (4-VGP). The production titers of 4-VPP and 4-VGP reached 48 and 33 mg/L at the maximum, respectively. Feeding experiments with 4-vinylphenol (4-VP), 4-vinylguaiacol (4-VG), and their glucosides indicated that 4-VPP and 4-VGP are formed by sequential glycosylation of 4-VP and 4-VG via their corresponding glucosides. Our results demonstrate the versatility of Pn cells for producing styrene derivatives, and indicate the presence of a unique glycosylation pathway to produce 4-VPP and 4-VGP in Pn cells.

DESign of Sustainable One-Pot Chemoenzymatic Organic Transformations in Deep Eutectic Solvents for the Synthesis of 1,2-Disubstituted Aromatic Olefins.

The self-assembly of styrene-type olefins into the corresponding stilbenes was conveniently performed in the Deep Eutectic Solvent (DES) mixture 1ChCl/2Gly under air and in the absence of hazardous organic co-solvents using a one-pot chemo-biocatalytic route. Here, an enzymatic decarboxylation of p-hydroxycinnamic acids sequentially followed by a ruthenium-catalyzed metathesis of olefins has been investigated in DES. Moreover, and to extend the design of chemoenzymatic processes in DESs, we also coupled the aforementioned enzymatic decarboxylation reaction to now concomitant Pd-catalyzed Heck-type C-C coupling to produce biaryl derivatives under environmentally friendly reaction conditions.

Phenolic acid decarboxylase of Aspergillus luchuensis plays a crucial role in 4-vinylguaiacol production during awamori brewing.

Aspergillus luchuensis has been used to produce awamori, a distilled liquor, in Okinawa, Japan. Vanillin, derived from ferulic acid (FA) in rice grains, is one of the characteristic flavors in aged and matured awamori, known as kusu. Decarboxylation of FA leads to the production of 4-vinylguaiacol (4-VG), which is converted to vanillin by natural oxidization. However, the mechanism underlying FA conversion to 4-VG has remained unknown in awamori brewing. In our previous studies, we showed that phenolic acid decarboxylase from A. luchuensis (AlPAD) could catalyze the conversion of FA to 4-VG, and that AlPAD is functionally expressed during koji making (Maeda et al., J. Biosci. Bioeng., 126, 162-168, 2018). In this study, to understand the contribution of AlPAD to 4-VG production in awamori brewing, we created an alpad disruptant (Δalpad) and compared its 4-VG productivity to that of the wild-type strain. The amount of 4-VG in the distillate of moromi prepared with the wild-type strain showed a significant increase, proportional to the time required for koji making. In the Δalpad strain, the amount of 4-VG was very small and remained unchanged during the koji making. In an awamori brewing test using koji harvested 42-66 h after inoculation, the contribution of AlPAD to 4-VG production was in the range of 88-94 %. These results indicate that AlPAD plays a key role in 4-VG production during awamori brewing.

A Pyrus communis gene for p-hydroxystyrene biosynthesis, has a role in defense against the pear psylla Cacopsylla biden.

Styrene analogs are known to be naturally synthesized in the leaves of pears and in other plant species, including several trees in the Styracaceae family. Styrene analogs are potential contributors to the aroma of wine, perfumes, pharmaceuticals, and other fermented foods and beverages. In addition, styrene analogs perform important ecological functions such as insecticidal and antifeedant activities against insects. We showed here that exogenous applications of styrene and p-hydroxystyrene caused a dramatic reduction the number of eggs laid by psylla and of subsequent nymph survival. Despite their importance specific reactions that lead to the biosynthesis of the styrene analogs in pear are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for pear genes with significant sequence similarity to bacterial phenolic acid decarboxylase. Herein described are the isolation and characterization of a pear phenolic acid decarboxylase, designated PyPAD1, which catalyzed the decarboxylation of p-coumaric acid and ferulic acid to p-hydroxystyrene and 3-methoxy-4-hydroxystyrene respectively. Its apparent Km values for p-coumaric acid and ferulic acid were 34.42 and 84.64 µM, respectively. The PyPAD1 preferred p-coumaric acid to ferulic acid as a substrate by a factor of 2.4 when comparing catalytic efficiencies in vitro. Expression analysis of PyPAD1 showed that the gene was transcribed in all five pear genotypes examined. However, transcript abundance was increased in correlation with the presence of p-hydroxystyrene in resistant cultivars Py-701 and Py-760 and in the sensitive cultivar Spadona when grafted on these resistant cultivars. Thus, PyPAD1 appears to be responsible for the decarboxylation of the p-coumaric acid, and for the production of metabolites that are active against pear psylla.

A Phenolic Acid Decarboxylase-Based All-Enzyme Hydrogel for Flow Reactor Technology.

Carrier-free enzyme immobilization techniques are an important development in the field of efficient and streamlined continuous synthetic processes using microreactors. Here, the use of monolithic, self-assembling all-enzyme hydrogels is expanded to phenolic acid decarboxylases. This provides access to the continuous flow production of p-hydroxystyrene from p-coumaric acid for more than 10 h with conversions ≥98% and space time yields of 57.7 g·(d·L)-1. Furthermore, modulation of the degree of crosslinking in the hydrogels resulted in a defined variation of the rheological behavior in terms of elasticity and mesh size of the corresponding materials. This work is addressing the demand of sustainable strategies for defunctionalization of renewable feedstocks.

Bioproduction of High-Concentration 4-Vinylguaiacol Using Whole-Cell Catalysis Harboring an Organic Solvent-Tolerant Phenolic Acid Decarboxylase From Bacillus atrophaeus.

The compound 4-vinyl guaiacol (4-VG) is highly valued and widely applied in the pharmaceutical, cosmetic, and food industries. The bioproduction of 4-VG from ferulic acid (FA) by non-oxidative decarboxylation using phenolic acid decarboxylases is promising but has been hampered by low conversion yields and final product concentrations due to the toxicities of 4-VG and FA. In the current study, a new phenolic acid decarboxylase (BaPAD) was characterized from Bacillus atrophaeus. The BaPAD possessed excellent catalytic activity and stability in various organic solvents. Whole Escherichia coli cells harboring intracellular BaPAD exhibited greater tolerances to FA and 4-VG than those of free BaPAD. A highly efficient aqueous-organic biphasic system was established using 1-octanol as the optimal organic phase for whole-cell catalysis. In this system, a very high concentration (1580 mM, 237.3 g/L) of 4-VG was achieved in a 2 L working volume bioreactor, and the molar conversion yield and productivity reached 98.9% and 18.3 g/L/h in 13 h, respectively.

Characterization and induction of phenolic acid decarboxylase from Aspergillus luchuensis.

Awamori is a traditional distilled liquor in the Ryukyu Islands, made from steamed rice by the action of the black-koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae. One of the specific flavors in aged awamori kusu is vanillin, which is derived from ferulic acid (FA) in rice grains. FA is released from the cell wall material in the rice grain by ferulic acid esterase produced by A. luchuensis. Through decarboxylation of FA, 4-vinylguaiacol (4-VG) is produced, which is transferred to the distilled liquor, and converted to vanillin by natural oxidization during the aging process. However, the actual mechanism for conversion of FA to 4-VG in the awamori brewing process is unknown. A genetic sequence having homology to the phenolic acid decarboxylase (PAD)-encoding region from bacteria and the yeast Candida guilliermondii has been identified in A. luchuensis mut. kawachii. In the present study, recombinant PAD from A. luchuensis, designated as AlPAD, expressed as a homodimer, catalyzed the conversion of FA to 4-VG, displayed optimal catalytic activity at pH 5.7 and 40°C, and was stable up to 50°C. Both rice bran and FA could induce the bioconversion of FA to 4-VG and the expression of AlPAD in A. luchuensis. The amount of AlPAD determined using western blotting correlated with the level of FA decarboxylase activity during koji production. In awamori brewing process, AlPAD might be responsible for a part of the conversion of FA to 4-VG.

4-Vinylphenol biosynthesis from cellulose as the sole carbon source using phenolic acid decarboxylase- and tyrosine ammonia lyase-expressing Streptomyces lividans.

Streptomyces lividans was adopted as a host strain for 4-vinylphenol (4VPh) production directly from cellulose. In order to obtain novel phenolic acid decarboxylase (PAD) expressed in S. lividans, PADs distributed among Streptomyces species were screened. Three novel PADs, derived from Streptomycessviceus, Streptomyceshygroscopicus, and Streptomycescattleya, were successfully obtained and expressed in S. lividans. S. sviceus PAD (SsPAD) could convert p-hydroxycinnamic acid (pHCA) to 4VPh more efficiently than the others both in vitro and in vivo. For 4VPh production directly from cellulose, l-tyrosine ammonia lyase derived from Rhodobacter sphaeroides and SsPAD were introduced into endoglucanase-secreting S. lividans, and the 4VPh biosynthetic pathway was constructed therein. The created transformants successfully produced 4VPh directly from cellulose.

Theoretical study of the reaction mechanism of phenolic acid decarboxylase.

The cofactor-free phenolic acid decarboxylases (PADs) catalyze the non-oxidative decarboxylation of phenolic acids to their corresponding p-vinyl derivatives. Phenolic acids are toxic to some organisms, and a number of them have evolved the ability to transform these compounds, including PAD-catalyzed reactions. Since the vinyl derivative products can be used as polymer precursors and are also of interest in the food-processing industry, PADs might have potential applications as biocatalysts. We have investigated the detailed reaction mechanism of PAD from Bacillus subtilis using quantum chemical methodology. A number of different mechanistic scenarios have been considered and evaluated on the basis of their energy profiles. The calculations support a mechanism in which a quinone methide intermediate is formed by protonation of the substrate double bond, followed by C-C bond cleavage. A different substrate orientation in the active site is suggested compared to the literature proposal. This suggestion is analogous to other enzymes with p-hydroxylated aromatic compounds as substrates, such as hydroxycinnamoyl-CoA hydratase-lyase and vanillyl alcohol oxidase. Furthermore, on the basis of the calculations, a different active site residue compared to previous proposals is suggested to act as the general acid in the reaction. The mechanism put forward here is consistent with the available mutagenesis experiments and the calculated energy barrier is in agreement with measured rate constants. The detailed mechanistic understanding developed here might be extended to other members of the family of PAD-type enzymes. It could also be useful to rationalize the recently developed alternative promiscuous reactivities of these enzymes.

Production of volatile phenols by kimchi Lactobacillus plantarum isolates and factors influencing their phenolic acid decarboxylase gene expression profiles.

Potential of kimchi lactic acid bacteria (LAB) isolates to produce volatile phenols and factors affecting their phenolic acid decarboxylase (padA) gene expression profiles were investigated in this study. Twelve percent (12%) of 50 tested LAB isolates were found to decarboxylate hydroxycinnamic acids. All six isolates were identified as Lactobacillus plantarum and possessed the padA gene. The highest padA expression was achieved on the third day of incubation with ferulic acid, with a relative expression of 3.30±0.32. The effects of glucose, substrate, and product concentrations, and the pH of the medium were investigated using response surface methodology for the first time in this study. The expression profiles of the padA gene were diverse in various stress environments. The concentration of p-coumaric acid was the most significant factor being positively correlated with the expression levels of the padA gene, but other factors did not show any significant effects. High concentrations of substrates could confer antibacterial activity. Therefore, decarboxylation reaction was suggested as a bacterial response to overcome the antibacterial activity. The phenolic acid decarboxylase activities of L. plantarum isolates found in this study can provide insights for their potential application in the development of food-grade flavors and additives.

Plectranthus amboinicus leaves stimulate growth of probiotic L. plantarum: evidence for ethnobotanical use in diarrhea.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves of Plectranthus amboinicus are consumed in India along with buttermilk (a probiotic source) during pathogen induced diarrhea. This treatment is known to reduce the number of episodes as well as duration of diarrhea. AIM OF THE STUDY: In the background of its ethnobotanical use, the present investigation was carried out to determine whether, apart from having an antimicrobial activity on pathogens, the leaves could possibly also have a positive effect on the beneficial microflora of the gut resulting in accelerated microbial ecological balance. MATERIALS AND METHODS: The growth stimulating activity of the hot water extract (HWE) of P. amboinicus leaves on probiotic Lactobacillus plantarum was determined by microbroth dilution technique and viable plate count method in selective medium (MRS) as well as in fermented milk. The ability of the bacteria to utilize the phytoconstituents of HWE primarily phenolic acids and sugars was determined by assaying for phenolic acid decarboxylase by SDS-PAGE and ß-galactosidase activity by ß-gal ONPG assay. RESULTS: HWE of P. amboinicus leaves inhibited growth of pathogens (Escherichia coli and Salmonella typhimurium) while stimulated the growth of L. plantarum. SDS-PAGE gel showed the presence of phenolic acid decarboxylase enzyme induced in the presence of HWE in L. plantarum indicating the utilization of polyphenols by the bacteria. Cells grown on HWE also showed ß-galactosidase activity indicating their ability to utilize sugars present in HWE. CONCLUSION: Indian borage leaves have a prebiotic effect on the probiotic bacteria (L. plantarum) which utilizes the phytoconstituents of the leaves by producing necessary metabolic enzymes. This work provides evidence in the traditional use of the leaves in the alleviation of diarrhea by accelerating microbial gut balance during infection.