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

120

101

Cinnamic acids (i.e., ferulic and caffeic acids) that are esterified to the vegetable cell walls should be enzymatically released to be absorbed in a mammal's intestines. A low dosage of ferulic acid in rodent diets stimulates insulin production and alleviates symptoms caused by diabetes (M. Sri Balasubashini, R. Rukkumani, and V. P. Menon, Acta Diabetol. 40:118-122, 2003). Several lactic acid bacteria are able to display ferulic acid esterase (FAE) activity, suggesting that their probiotic activity could be, in part, mediated by the slow release of ferulic acid. In the present work, we describe the isolation of one strain identified as being Lactobacillus johnsonii that displayed strong FAE activity in stool samples from diabetes-resistant biobreeding rats. These animals are genetically susceptible to becoming diabetic but do not develop the disease. By using genomic analysis coupled to protein purification and catalytic screening, we were able to purify two proteins with FAE activity. The enzymes displayed 42% sequence identity and a broad range of substrate preferences. High affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate (K m ‫؍‬ 20 to 60 M) and chlorogenic acid (K m ‫؍‬ 10 to 50 M) were observed. The strain isolated herein as well as the enzymes studied could be potentially useful for the formulation of probiotics to ameliorate diabetes symptoms.According to the Centers for Disease Control and Prevention (CDC), approximately 23.6 million Americans suffer from diabetes. In the year 2000, this metabolic disease was directly or indirectly responsible for 200,000 deaths. On average, diabetes costs more than 170 billion dollars annually to the American economy. Diets abundant in carbohydrates, increasing rates of obesity, and sedentary lifestyles in combination with genetic predisposition are key factors associated with the development of diabetes. In addition to these environmental factors, notable changes in the composition of the gut microbial ecosystem of diabetic patients were recently described (36).A clever analysis utilized to measure microbially diverse populations based on genomic principles reported previously by Furrie (17) was used by Roesch and coworkers (30) to prove that rodents with diabetes have a specifically associated gut microflora. A deep characterization of the intestinal microflora of biobreeding diabetes-resistant (BB-DR) rats showed that the presence of Lactobacillus reuteri, Lactobacillus johnsonii, and Bifidobacterium species is predominant, compared with the bacterial species observed in biobreeding diabetes-prone (BB-DP) rats (30). In agreement with the large presence of probiotic-like bacteria, it was previously reported that the oral administration of lactic acid bacteria (LAB) can help to reduce blood glucose levels (25-27, 40) by stimulating insulin secretion via changes in autonomic neurotransmission (34). However, it is not clear how LAB species are able to induce these changes. Neither genetic nor physiological characteristics of the st...

Section: Vol 75 2009 Cinnamoyl Esterase From Lactobacillus Johnsonisupporting

confidence: 66%

Biochemical Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated from Stool Samples of Diabetes-Resistant Rats

Lai

Lorca

González

2009

120

101

“…These results demonstrated that UmChlE differed from other chlorogenic acid esterases described in the literature. While Asther et al (13) and Adachi et al (15) identified the native chlorogenic acid esterases as homodimers with subunits of about 80 kDa each, Benoit et al (14) suggested that the recombinant A. niger enzyme exists in a tetrameric form. Schöbel and Pollmann (11) also characterized the second known chlorogenic acid esterase from A. niger as a tetramer (four 60-kDa subunits), whereas Okamura and Watanabe (16) described the hydroxycinnamic acid ester hydrolase of A. japonicus as a monomer of 145 kDa.…”

Section: Resultsmentioning

confidence: 99%

“…The determined temperature optimum was lower than those reported previously for chlorogenic acid esterases and most of the feruloyl esterases (36). Furthermore, the purified enzyme was less thermostable than those previously described (11,13,14). The applicability of UmChlE for ester synthesis was examined by using 1:1 (vol/vol) buffer emulsions with different organic solvents (Table 2).…”

mentioning

confidence: 99%

“…In 1980, Schöbel and Pollmann (11,12) reported a specific chlorogenic acid esterase from a pectinolytic enzyme preparation of Aspergillus niger. More recently, Asther et al (13) demonstrated that another A. niger enzyme releases caffeic acid from industrial by-products such as coffee pulp (CP) and apple marc. The corresponding gene was homologously overexpressed by Benoit et al (14), and the properties of the recombinant and native enzymes were compared.…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

Nieter

Haase-Aschoff

Kelle

et al. 2015

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ϳ71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic , respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. P lant cell walls consist of a heterogeneous mixture of polymers, including cellulose, hemicellulose, pectin, and lignin. In addition to these main components, hydroxycinnamates play a key role in the formation of a complex network of these polymers. While hydroxycinnamates covalently cross-link plant cell wall polysaccharides to each other by ester bonds, links between polysaccharides and lignin are formed mainly by ether linkages (1). Thus, these phenolic acids (mainly ferulic, p-coumaric, and caffeic acids) not only influence the rigidity and mechanical properties of the cell wall but also play a role in plant defense (2). They are linked differently to sugar residues in plants: in grasses such as wheat, barley, or maize, ferulates and p-coumarates are mainly found esterified to the O-5 position of arabinose residues, whereas in pectin of dicotyledons, such as spinach or sugar beet, ferulic acid is esterified to the O-2 position of arabinose and the O-6 position of galactose residues (3). Furthermore, hydroxycinnamates also exist as soluble ester conjugates of quinic acid, the best-known example being chlorogenic acid [3-(3,4-dihydroxycinnamoyl)quinic acid] (4). Particularly high concentrations of chlorogenic acid are present in coffee, apple, pear, and potato tuber (4). The health, cosmetic, and pharmaceutical industries are especially interested in hydroxycinnamates due to their anticarcinogenic, anti-inflammatory, and antioxidant properties (5). Consequently, enzymes that are able to release these phenolic compounds are not only essential for the degradation of plant biomass but also of interest for potential food and medical applications of the released products.One group of these enzymes is the feruloyl esterases (FAEs) (EC 3.1.1.73), a subclass of the carboxylic ester hydrolases (EC 3.1.1). They hydrolyze the ester lin...

“…Overlapping substrate specificities were also observed for FAE-B of A. niger, an enzyme that converted different esters, including feruloylated oligosaccharides, wheat arabinoxylan, and alky-aryl esters; interestingly, this enzyme showed significant sequence similarity to an Aspergillus tannase (EC 3.1.1.20) but without possessing the respective enzymatic activity (6). A chlorogenic acid hydrolase (EC 3.1.1.42) with broad substrate specificity was described for another A. niger strain and suggested to be a new esterase/protein type, since it did not show any homology to known cinnamoyl esterases (2). Eventually, Koseki et al (2010) described an FAE from A. kawachii that contains the carbohydrate-binding module from GH family 54 (␣-L-arabinofuranosidase, EC 3.2.1.55) (27).…”

Section: Figmentioning

confidence: 95%

The Wood Rot Ascomycete Xylaria polymorpha Produces a Novel GH78 Glycoside Hydrolase That Exhibits α- l -Rhamnosidase and Feruloyl Esterase Activities and Releases Hydroxycinnamic Acids from Lignocelluloses

Nghị

Bittner

Kellner

et al. 2012

ABSTRACTSoft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g−1) or beech wood (up to 80 mU g−1). Following the ester-cleaving activity toward methyl ferulate, a hydrolase ofXylaria polymorphawas produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg−1). The peptide sequence of the purified protein deduced from the gene sequence and verified byde novopeptide sequencing shows high similarity to putative α-l-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-l-rhamnopyranoside and α-l-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, orp-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest thatX. polymorphaGH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses.