Han-Dae Yun

Phylogenetic analysis of protozoa in the rumen contents of cow based on the 18S rDNA sequences

Aims:  To examine the diversity of protozoa in the rumen contents of cow.

Changes in the activity of the multifunctional β-glycosyl hydrolase (Cel44C-Man26A) from Paenibacillus polymyxa by removal of the C-terminal region to minimum size

Paenibacillus polymyxa GS01 secretes Cel44C-Man26A as a multifunctional enzyme with cellulase, xylanase, lichenase, and mannanase activities. Cel44C-Man26A consists of 1,352 amino acids in which present a catalytic domain (CD) of the glycosyl hydrolase family 44 (GH44), fibronectin domain type 3 (Fn3), catalytic domain of glycosyl hydrolase family 26 (GH26), and a cellulose-binding module type 3 (CBM3). A truncated Cel44C-Man26A protein, consisting of 549 amino acid residues, reacted as a multifunctional mature enzyme despite the absence of the 10 amino acids containing GH44, Fn3, GH26, and CBM3. However, the multifunctional activity was not found in the mature Cel44C-Man26A protein truncated to less than 548 amino acids. The truncated Cel44C-Man26A proteins showed the optimum pH for the lichenase activity was pH 7.0, pH 6.0 for the xylanase and mannanase, and pH 5.0 for the cellulase. The truncated Cel44C-Man26A proteins exhibited enzymatic activity 40–120% higher than the full-length Cel44C.

Cloning and characterization of cel8A gene from Rhizobium leguminosarum bv. trifolii 1536

Aims:  To isolate the cellulase gene from Rhizobium leguminosarum bv. trifolii 1536.

Phylogenetic analysis of yeast in the rumen contents of cattle based on the 26S rDNA sequence

The composition of yeast communities in the rumen of cattle was investigated using comparative DNA sequence analysis of yeast 26S rDNA genes. 26S rDNA libraries were constructed from rumen fluid (FF), rumen solid (FS) and rumen epithelium (FE). A total of 97 clones, containing a partial 26S rDNA sequence of 0.6 kb length, were sequenced and subjected to an on-line similarity search. The 41 FF clones could be divided into five classes. The largest class was affiliated with Pezizomycotina class (85.4% of clones), and the remaining classes were related with the Urediniomycotina (2.4%), Hymenomycetes (4.9%), Ustilaginomycetes (4.9%) and Saccharomycotina (2.4%) classes. The 26 FE clones could be divided into three classes and the Saccharomycetes class (92.4 % of clones) was the largest group. The remaining classes were related with either Pezizomycotina (3.8%) or Ustilaginomycetes (3.8 %). The 30 FS clones were all affiliated with Saccharomycotina. Saccharomycotina were predominant in rumen epithelium and rumen solid while Pezizomycotina were predominant in rumen fluid. Yeast belonging to the Saccharomycotina class was predominant in the rumen as a whole (57%). One clone (FF34) had less than 90% similarity to any sequence in the database and was thus apparently unrelated to any previously described yeast.

Cloning and Identification of Essential Residues for Thermostable β-glucosidase (BgIB) from Thermotoga maritima

A hyperthermophilic bacterium Thermotoga maritima produced thermostable β-glucosidase. The gene encoding β-glucosidase from T. maritima MSB8 was cloned and expressed in Escherichia coli. The enzyme (BglB) hydrolyzed β-glucosidic linkages between glucose and alkyl, aryl of saccharide groups such as salicin, arbutin, and ρNPG. The insert DNA contained ORF with 2,166 bp encodes a 721 amino acids (calculated molecular mass of 80,964 and pI of 4.93). The amino acid sequence of BglB showed the similarity to family 3 glycosyl hydrolases. The molecular weight of the enzyme was estimated to be approximately 81 kDa by MUG-nondenaturing PAGE (4-methylumbelliferyl β-D-glucoside-nondenaturing polyacrylamide gel electophoresis) and SDS-PAGE. The β-glucosidase exhibited maximal activity at pH 7.0 and 80°C. By exchanging two possible residues (Glu-232 and Asp-242) to Ala by site-directed mutagenesis method, it was found that these were essential for enzymatic activity.

Effects of Rhodobacter sp. SA16 on Lettuce (Lactuca sativa L.) in Plastic Film House

This experiment was conducted to determine the development of mixed organic fertilizer using photosynthetic bacteria and mass production of mixed microbial compound for the environment -friendly agriculture. Photosynthetic bacteria, Rhodobacter sp. SA16 was isolated from soil collected by plastic film house. The SA16 strain was identified based on 16S rDNA sequence analysis and it is closely related to Rhodobacter sp. (100% similarity). The mixed organic fertilizer using SA16 was made of N-P2O5-K2O=60-10-20 g kg -1 with combined soybean cake, sesame cake, powdered blood, fish meal, powdered bones and red-yellow soil. The mixed organic fertilizer 0.45, 0.90 and 1.35 Mg ha -1 application in Ihyeon series was treated based on soil testing for lettuce cultivation in plastic film house. These results showed that the yield was increased the 18 and 19% over control by the mixed organic fertilizer application 0.45 and 0.90 Mg ha -1 , respectively. In the physical properties of the soil, the porosity of mixed organic fertilizer 1.35 Mg ha -1 treatment was highest at 58.8%. Our results clearly revealed that the organic fertilizer using Rhodobacter sp. SA16 and mass production of mixed strains could be a useful technology in pursuing environment-friendly agriculture.