Takuya Koseki

Occurrence, properties, and applications of feruloyl esterases

Feruloyl esterases hydrolyze the ester linkages of ferulic and diferulic acids present in plant cell walls. This interesting group of enzymes also has a potentially broad range of applications in the pharmaceutical and agri-food industries. An overview of the current knowledge of fungal feruloyl esterases focusing on the diverse of substrate specificity and potential applications is presented in this review. Furthermore, biological functions of ferulic acid are discussed.

Novel Effects of a Single Administration of Ferulic Acid on the Regulation of Blood Pressure and the Hepatic Lipid Metabolic Profile in Stroke-Prone Spontaneously Hypertensive Rats

We studied the effects of a single oral administration of ferulic acid (FA) on the blood pressure (BP) and lipid profile in stroke-prone spontaneously hypertensive rats (SHRSP). Male 12-week-old SHRSP were administered FA (9.5 mg/kg of body weight) and distilled water as the control (C) (1 mL) via a gastric tube. The hypotensive effect of FA was observed at the lowest value after 2 h administration. A decrease in the angiotensin-1-converting enzyme (ACE) activity in the plasma corresponded well with the reduction of BP. Plasma total cholesterol and triglyceride levels were lower after 2 h administration. The mRNA expression of genes involved in lipid and drug metabolism was downregulated in the FA group. These results suggest that oral administration of FA appears beneficial in improving hypertension and hyperlipidemia.

Characterization of two distinct feruloyl esterases, AoFaeB and AoFaeC, from Aspergillus oryzae

Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0–10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid.

Cloning of the xynNB Gene Encoding Xylanase B from Aspergillus niger and Its Expression in Aspergillus kawachii

Abstract Aspergillus niger IFO 4066 produced two xylanases, xylanase A (XynNA) and xylanase B (XynNB), in culture medium, and these enzymes were purified. Acidophilic xylanase such as xylanase C (XynC) of white koji mold (Aspergillus kawachii IFO 4308) was not detected in A. niger cultures. However, results of Southern analysis using xynC cDNA of A. kawachii as a probe suggested that A. niger contained a gene homologous to xynC of A. kawachii. Therefore, we cloned this xylanase gene from A. niger. The predicted amino acid sequence of the cloned xylanase showed a homology to that of xynC of A. kawachii. However, a large number of amino acid substitutions were detected, especially in the N-terminal region. Both this cloned gene and xynC gene of A. kawachii had an intron at the same position in the coding region. The cloned gene was expressed in A. kawachii and a large quantity of xylanase was produced. The elution profile on an anion exchange chromatogram and the N-terminal amino acid sequence of the xylanase purified from the transformant were the same as those of XynNB. This confirmed that the cloned gene encoded XynNB.

Structural characteristics, properties, and in vitro digestibility of rice

ABSTRACT Using rice samples derived from normal rice cultivars and endosperm starch mutant, we investigated key factors contributing to the enzyme digestibility of steamed rice grains. The chemical composition of polished rice grains, structural features of endosperm starch, and enzyme digestibility of steamed rice grains were examined. The protein content of polished rice grains was 4.6–9.1%, amylose content was 4–27%, the DPn of purified amylose was 900–1,600, the amylopectin short/long chain ratio was 1.2–5.9, and the enzyme digestibilities of steamed polished rice grains were 0.9–12.6 °Brix. Amylose content and RVA parameters (viscosity, breakdown, and setback) correlated significantly with enzyme digestibility of steamed rice grains. Multiple regression formulas were constructed to predict digestibility of steamed rice grain as a function of the molecular characteristics of the starch. When both amylose content and the short/long chain amylopectin ratio were used as predictor variables, they accounte...

Conversion of ferulic acid into 4-vinylguaiacol, vanillin and vanillic acid in model solutions of shochu

4-Vinylguaiacol (4-hydroxy-3-methoxystyrene), vanillin (4-hydroxy-3-methoxybenzaldehyde), and vanillic acid (4-hydroxy-3-methoxybenzoic acid) were isolated from distilled and stored model solutions of shochu (MSS) that originally contained only ferulic acid (4-hydroxy-3-methoxycinnamic acid) using a solid-phase extraction technique. These compounds were analyzed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The amounts of the metabolites converted from ferulic acid was affected by pH, alcohol concentration, and temperature of during storage. The identities of the metabolites were tentatively determined by gas chromatography-mass spectrometry (GC-MS). The results of this experiment suggested that vanillin was formed via 4-vinylguaiacol from ferulic acid, and was then converted to vanillic acid. The pathway of the decarboxylation of ferulic acid appeared to be one of the chemical transformation.

Pyrrospirones A and B, apoptosis inducers in HL-60 cells, from an endophytic fungus, Neonectria ramulariae Wollenw KS-246

Pyrrospirones A and B have been isolated from unpolished rice cultures of the endophytic fungus Neonectria ramulariae Wollenw KS-246. Their absolute stereostructures (1 and 2) were elucidated through spectroscopic methods using 1D and 2D NMR techniques and chemical transformations, including the modified Moshers method. The compounds exhibited cytotoxicity and induced apoptosis in human promyelocytic leukemia HL-60 cells.

The family 42 carbohydrate-binding module of family 54 α-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose

Alpha-L-arabinofuranosidase catalyses the hydrolysis of the alpha-1,2-, alpha-1,3-, and alpha-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-alpha-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 10(3) M(-1). These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.

Role of Two α-l-arabinofuranosidases in arabinoxylan degradation and characteristics of the encoding genes from shochu koji molds, Aspergillus kawachii and Aspergillus awamori

Two different alpha-L-arabinofuranosidases from Aspergillus kawachii were purified and characterized. The two enzymes acted synergically with xylanase in the degradation of arabinoxylan and resulted in an increase in the amount of ferulic acid release by feruloyl esterase. Both enzymes were acidophilic and acid stable enzymes which had an optimum pH of 4.0 and were stable at pH 3.0-7.0. The general properties of the enzymes including pH optima and pH stability were similar to those of Aspergillus awamori. These results suggest that the alpha-L-arabinofuranosidases contribute to an increase in cereal utilization and formation of aroma in shochu brewing. Two different genes encoding alpha-L-arabinofuranosidases from A. kawachii, designated as AkabfA and AkabjB, and those from A. awamori, designated as AwabfA and AwabjB, were also cloned and characterized. The difference between the sequences of AkabfA and AwabfA was only one nucleotide, resulting in an amino acid difference in the sequence, and the enzymes were assigned to family 51 of glycoside hydrolases. On the other hand, the differences between the sequences of AkabjB and AwabjB and between their encoding proteins were two nucleotides and one amino acid residue, respectively, and the enzymes were assigned to family 54 of glycoside hydrolases. On comparison of the abfA and abjB genes among A. kawachii, A. awamori, and A. niger, the relationship between the two genes for A. kawachii and A. awamori was much closer than those between A. niger and the others. Northern analyses showed that transcription of AkabfB was greater than that of AkabfA in the presence of L-arabitol and L-arabinose, and that transcriptions of both genes were not induced in the presence of sucrose and glucose.

Fusaristatins A and B, two new cyclic lipopeptides from an endophytic Fusarium sp.

Two new cyclic lipopeptides, fusaristatins A (1) and B (2) were isolated from rice cultures of a Fusarium sp. YG-45 in the course of a screening of endophytic fungi. Their structures of 1 and 2 were determined by spectroscopic methods. 2 showed a moderate inhibitory effect on topoisomerases I (IC50: 73 μM) and II (IC50: 98 μM) without cleavable complexes. Furthermore, 1 and 2 showed the growth-inhibitory activity toward lung cancer cells LU 65 with IC50 values of 23 and 7 μM, respectively.