Haruhiko Kawasaki

Purification and properties of two type-B α-l-arabinofuranosidases produced by Penicillium chrysogenum

Two distinct extracellular alpha-L-arabinofuranosidases (AFases; EC 3.2.1.55) were purified from the culture filtrate of Penicillium chrysogenum 31B. The molecular masses of the enzymes were estimated to be 79 kDa (AFQ1) and 52 kDa (AFS1) by SDS-PAGE. Both enzymes had their highest activities at 50 degrees C and were stable up to 50 degrees C. Enzyme activities of AFQ1 and AFS1 were highest at pH 4.0 to 6.5 and pH 3.3 to 5.0, respectively. Addition of 10 mg/ml arabinose to the reaction mixture decreased the AFS1 activity but hardly affected AFQ1. Both enzymes displayed broad substrate specificities; they released arabinose from branched arabinan, debranched arabinan, arabinoxylan, arabinogalactan, and arabino-oligosaccharides. AFS1 also showed low activity towards p-nitrophenyl-beta-D-xylopyranoside. An exo-arabinanase, which catalyzes the release of arabinobiose from linear arabinan at the nonreducing terminus, acted synergistically with both enzymes to produce L-arabinose from branched arabinan.

Molecular characterization of a Penicillium chrysogenum exo‐1,5‐α‐L‐arabinanase that is structurally distinct from other arabinan‐degrading enzymes

The nucleotide sequence of the abnx cDNA gene, which encodes an exo‐arabinanase (Abnx) of Penicillium chrysogenum 31B, was determined. Abnx was found to be structurally distinct from known arabinan‐degrading enzymes based on its amino acid sequence and a hydrophobic cluster analysis. The protein in the protein database with the highest similarity to Abnx was the Neurospora crassa conserved hypothetical protein. The abnx cDNA gene product expressed in Escherichia coli catalyzed the release of arabinobiose from α‐1,5‐L‐arabinan. The activity of the recombinant Abnx towards a series of arabino‐oligosaccharides, as expressed by k cat/K m value, was greatest with arabinohexaose.

Molecular cloning and expression of a polygalacturonase gene in Saccharomyces cerevisiae

An endo-polygalacturonase gene was cloned from a mutant of Saccharomyces cerevisiae that can produce a polygalacturonase, and the nucleotide sequence was identified. The polygalacturonase gene consists of 1086 bp. When the polygalacturonase gene was introduced into a strain of S. cerevisiae that cannot produce a polygalacturonase, the transformant secreted a polygalacturonase identical to that produced by the polygalacturonase-producing mutant.

Purification, Characterization, and Overexpression of Thermophilic Pectate Lyase of Bacillus sp. RN1 Isolated from a Hot Spring in Thailand

A thermophilic pectate lyase, Pel SWU, was isolated from a culture filtrate of Bacillus sp. RN1 isolated from a hot spring in Ranong Province, Thailand. The enzyme was purified to homogeneity using cation-exchange and hydrophobic column chromatographies. The molecular mass of Pel SWU was estimated to be 33 kDa. The specific substrate was demethylated galacturonic acid. The enzyme was stable at pH 4.0–10.0 and at temperatures up to 70 °C in the presence of calcium and polygalacturonic acid (PGA). The optimum pH and temperature were 10.0 and 90 °C. The pel gene encoding Pel SWU was 1,023 bp, which corresponds to 341 amino acids. The properties of the recombinant enzyme was similar to those of Bacillus Pel SWU. Unsaturated di- and trigalacturonic acids were formed mainly as the final products of degradation by Pel SWU, as revealed by high-performance anion-exchange chromatography (HPAEC) and electrospray ionization mass spectrometry (ESI-MS) analyses. This thermophilic pectate lyase should be useful in the degradation of pectin networks at high temperature.

Enzymatic Synthesis of Hydroxycinnamic Acid Glycerol Esters Using Type A Feruloyl Esterase from Aspergillus niger

We found that hydroxycinnamic acid (HA) glycerol esters such as 1-sinapoyl glycerol and 1-p-coumaroyl glycerol can be synthesized through a direct esterification reaction using a type A feruloyl esterase from Aspergillus niger. The water solubilities of HA glycerol esters were higher than those of the original chemicals. HA glycerol esters absorbed ultraviolet light and scavenged 1,1-diphenyl-2-picrylhydrazyl radicals.

Acidic condition-inducible polygalacturonase of Aspergillus kawachii

Abstract Aspergillus kawachii IFO 4308, which can grow in an extremely acidic condition (pH 2), produced some extracellular polygalacturonases (PGase). However, pH 2 and pH 5 culture filtrates showed different pH PGase activity profiles. Anion exchange chromatographies revealed that the PGase compositions of the two culture filtrates were different, and dominant enzymes (PGase-A1 and -A2 in the pH 2 culture and PGase-B in the pH 5 culture) were purified and characterized. The optimal pH was pH 4 for A1, pH 3 for A2, and pH 5 for B. PGase-A1 and -A2 were more stable at low pH than PGase-B. Molecular masses of PGase-A1, -A2, and -B were 43, 83, and 71 kDa, respectively. The N-terminal amino acid sequence of PGase-B was similar to those of other fungal PGases, but distinctly different from those of PGase-A1 and -A2. These results suggest that PGase-A1 and -A2 may be acidic condition-inducible enzymes and that a pH-regulated expression system is involved in the PGase production of A. kawachii .

Cloning and heterologous expression of gene encoding A polygalacturonase from Aspergillus awamori.

A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.

Characteristics of Wines Made by Saccharomyces Mutants Which Produce a Polygalacturonase under Wine-Making Conditions

Wines by yeast mutants producing polygalacturonase in high glucose concentration, from Saccharomyces wine-making strains, had higher filterability and more concentrated anthocyanin contents than that of their parent strains. These results suggest that the clarification process was improved at a lower cost by the low viscosity and that high-quality wines result from the increase in the anthocyanin contents.

Isolation and expression of the gene which encodes a novel enzyme with polymethoxygalacturonate-degrading activity in Trichosporon penicillatum.

The novel gene named PSX1, encoding a new protopectinase with the polymethoxygalacturonase activity, was isolated from Trichosporon penicillatum. Nucleotide sequencing revealed that the PSX1 gene is composed of 1080 bases (360 amino acids, 38 747 Da). The N‐terminal amino acid sequences of the open reading frame correspond to a signal peptide and propeptide processed by a Kex2‐like proteinase. Mature PPase SX1 was composed of 334 amino acids (36 121 Da). PPase SX1 produced by a S. cerevisiae transformant harboring the PSX1 gene degraded methoxylated polygalacturonic acid as a substrate, but not degraded unmethoxylated polygalacturonic acid.