Yong-Suk Lee

Hydrolysis of isoflavone glycoside by immobilization of β-glucosidase on a chitosan-carbon in two-phase system

We explored a method to examine the hydrolysis of isoflavone glycoside by immobilizing β-glucosidase on chitosan-carbon beads in an aqueous-organic two-phase system. The chitosan-carbon beads were cross-linked with glutaraldehyde to immobilize β-glucosidase from Exiguobacterium sp. DAU5. The optimal pH and temperature were 9.0 and 55 °C, respectively. Under the optimized conditions, crude and purified enzymes immobilized onto chitosan-carbon beads gave yields of 16.7% and 60%, respectively. The specific activities of immobilized crude and purified enzymes were 4.3 U/g and 6 U/g, respectively. The immobilized enzyme retained more than 80% of its maximum activity at pH 7.0-11.0, while temperature was more influential (80% activity after 40 °C for 1.5 h, but only 40% activity after 55 °C for 0.5 h, respectively. The immobilized enzyme was able to hydrolyze isoflavone glycoside in an aqueous-organic two-phase system, and the hydrolyzed products were enriched in the organic phase, making it easy to recover the products, i.e., genistein and daidein from the reaction system. These results suggest that immobilized β-glucosidase may be applicable for the industrial-scale hydrolysis of isoflavone glycoside.

Recombinant Expression and Characterization of an Organic-Solvent-Tolerant α-Amylase from Exiguobacterium sp. DAU5

The enzyme from halophilic microorganisms often has unique properties such as organic-solvent-tolerance. In this study, a novel organic-solvent-tolerant α-amylase gene was cloned from the mild halophile Exiguobacterium sp. DAU5. The open reading frame (ORF) of the enzyme consisted of 1,545xa0bp and encoded 514 amino acids, the primary sequence revealed that it belongs to the glycoside hydrolase (GH) family 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an AmyH monomer of 57xa0kDa. The enzyme exhibited maximal activity at 40xa0°C in pHu20098.5 glycine–NaOH buffer, and the activity was strongly inhibited by Zn2+, Cu2+, and Fe2+. The α-amylase AmyH exhibited high hydrolysis activity toward soluble starch, and the major hydrolysis products were maltose, maltotriose, and maltopentaose; the AmyH could not efficiently hydrolyze oligosaccharides smaller than maltoheptaose, nor could it act on the β-1,4 or α-1,6 glucosidic bonds in xylan or pullulan, respectively. In addition, the α-amylase exhibited better tolerance to organic solvents, as it was stable in the presence of dimethylsulfoxide (DMSO), methanol, ethanol, and acetone. Base on all of these results, the enzyme could be useful for practical application in the bakery industry and in biotechnological processes that occur in the presence of organic solvents.

Characterization of a cold-active β-glucosidase from Paenibacillus xylanilyticus KJ-03 capable of hydrolyzing isoflavones daidzin and genistin.

Paenibacillus xylanilyticus KJ-03 isolated from konjac field, showed β-glucosidase activity on tryptic soy agar plate supplemented with 0.1xa0% esculin and 0.25xa0% ferric ammonium citrate. A genome library was constructed to obtain the β-glucosidase gene and a recombinant clone, pGlc2-3 was selected. The 2,247xa0bp gene encoding KJ-03 β-glucosidase consisted of 749 amino acids. The deduced amino acids of BglA were 61xa0% homologous with that of the β-glucosidase from Bacillus cereus AH1272, which belongs to the glycoside hydrolase family 3. His-tagged β-glucosidase was purified by using His-Trap column and characterized. KJ-03 β-glucosidase was showed as a single band with about 82xa0kDa on SDS-PAGE. The purified enzyme has optimal activity at 20xa0°C and pH 7.0 using p-NPβG and 72xa0% of the maximal activity was still remaining at 10xa0°C. The β-glucosidase has optimal activity at low temperatures indicating that it is a cold-active enzyme. The substrate specificity showed that the purified enzyme hydrolyzed aryl β-glucoside substrates and isoflavones such as daidzin and genistin.

Isolation and Characterization of a Novel Cold-Adapted Esterase, MtEst45, from Microbulbifer thermotolerans DAU221

A novel esterase, MtEst45, was isolated from a fosmid genomic library of Microbulbifer thermotolerans DAU221. The encoding gene is predicted to have a mass of 45,564 Da and encodes 495 amino acids, excluding a 21 amino acid signal peptide. MtEst45 showed a low amino acid identity (approximately 23–24%) compared with other lipolytic enzymes belonging to Family III, a closely related bacterial lipolytic enzyme family. MtEst45 also showed a conserved GXSXG motif, G131IS133YG135, which was reported as active site of known lipolytic enzymes, and the putative catalytic triad composed of D237 and H265. Because these mutants of MtEst45, which was S133A, D237N, and H265L, had no activity, these catalytic triad is deemed essential for the enzyme catalysis. MtEst45 was overexpressed in Escherichia coli BL21 (DE3) and purified via His-tag affinity chromatography. The optimal pH and temperature of MtEst45 were estimated to be 8.17 and 46.27°C by response surface methodology, respectively. Additionally, MtEst45 was also active between 1 and 15°C. The optimal hydrolysis substrate for MtEst45 among p-nitrophenyl esters (C2–C18) was p-nitrophenyl butyrate, and the Km and Vmax values were 0.0998 mM and 550 μmol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg2+, Zn2+, and Cu2+ ions; by phenylmethanesulfonyl fluoride; and by β-mercaptoethanol. Ca2+ did not affect the enzymes activity. These biochemical properties, sequence identity, and phylogenetic analysis suggest that MtEst45 represents a novel and valuable bacterial lipolytic enzyme family and is useful for biotechnological applications.

Characterization of maltotriose production by hydrolyzing of soluble starch with α-amylase from Microbulbifer thermotolerans DAU221

A maltotriose-producing α-amylase, AmyA, from a newly isolated bacterial strain Microbulbifer thermotolerans DAU221 was purified and characterized in the heterologous host, Escherichia coli, using the pCold I vector. The amyA gene encoded a 761-residue protein composed of a 33 amino acid secretion signal peptide. The purified α-amylase with a molecular mass of 80xa0kDa, approximately, shared a sequence motif characteristic of the glycoside hydrolase family 13. The enzyme was optimally active, at 50xa0°C in sodium phosphate buffer (pH 6.0), by the traditional one factor-at-a-time method. But the optimal conditions of time, temperature, and pH for production of maltotriose from soluble starch were 1.76xa0h, 44.95xa0°C, and pH 6.35 by response surface methodology, respectively. Maltotriose, as the major enzyme reaction product, was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The enzyme was found to be inhibited by the addition of 10xa0mM Cu2+, Fe3+, Hg2+, Zn2+, and EDTA, but exhibited extreme stability toward hexane. The Km and Vmax values for the hydrolysis of soluble starch were 1.08xa0mg/mL and 1.736xa0mmol maltotriose/mg protein/min, respectively.

β-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties

The gene encoding the cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) of Paenibacillus illinoisensis was isolated, cloned, sequenced and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34-residues. The deduced amino acid sequence of the CGTase from P. illinoisensis ZY-08 exhibited highest identity (99xa0%) to the CGTase sequence from Bacillus licheniformis (P14014). The four consensus regions of carbohydrate converting domain and Ca2+ binding domain could be identified in the sequence. The CGTase was purified by using cold expression vector, pCold I, and His-tag affinity chromatography. The molecular weight of the purified enzyme was about 74xa0kDa. The optimum temperature and pH of the enzyme were 40xa0°C and pH 7.4, respectively. The enzyme activity was increased by the addition of Ca2+ and inhibited by Ba2+, Cu2+, and Hg2+. The Km and Vmax values calculated were 0.48xa0mg/ml and 51.38xa0mg of β-cyclodextrin/ml/min. The ZY-08 and recombinant readily converted soluble starch to β-cyclodextrin but ZY-08 did not convert king oyster mushroom powder and enoki mushroom powder. However the recombinant CGTase converted king oyster mushroom powder and enoki mushroom powder to β-cyclodextrin.

Complete genome sequence and analysis of three kinds of β-agarase of Cellulophaga lytica DAU203 isolated from marine sediment

Cellulophaga lytica DAU203 (KACC 19187), isolated from the marine sediment in Korea, has a strong ability to degrade agar. The genome of C. lytica DAU203 contains a single chromosome that is 3,952,957bp in length, with 32.02% G+C contents. The genomic information predicted that the DAU203 has the potential to be utilized in various enzymatic industries.

Complete genome sequence of cold-adapted enzyme producing Microbulbifer thermotolerans DAU221

Microbulbifer thermotolerans DAU221 was preliminary isolated from the marine sediment samples in the Republic of Korea. Here, we present the complete genome sequence of M. thermotolerans DAU221, which consisted of 3,938,396 base pairs with a GC content of 56.57%. This genomic information should help us find the industrially useful enzymes.

Molecular functions of long noncoding transcripts in plants

Long noncoding RNAs (lncRNAs) are transcribed from the genomes of various eukaryotes. LncRNAs are potent regulators of various developmental processes and respond to external stimuli. Recent studies have shown that lncRNAs function as potent cis- and trans-regulators of gene expression and as modular scaffolds of chromatin-modifying complexes. Several plant lncRNAs transcribed in response to various external signals and stresses have been identified. However, only a few of these lncRNAs have been characterized, and information regarding their regulatory mechanisms is limited. Here, we describe our current understanding of the biological functions of plant lncRNAs.

Growth Promotion of Lettuce by Biofertilizer, BIOACTIVE, Prepared from Bacillus subtilus HR-1019 and N-acetyl-thioproline

A biofertilizer, BIOACTIVE, was manufactured by N-acetyl-thioproline (ATCA) and mineral phosphate solubilizing bacteria. The growth promoting effect of the biofertilizer on lettuce was evaluated under three different pot conditions, and its stability was assessed in the field. According to the results of the pot experiments, plant growth was improved compared with that of control: 128%, 122%, and 153% for the leaf number, leaf length, and leaf mass, respectively. Applying the manufactured biofertilizer increased the concentration of phosphate: 118% and 132% in the cultivation soil and plant cells, respectively. These show that BIOACTIVE may have potential as an effective biofertilizer in agriculture.