Isolation and Characteristics of Alkaline Protease Producing

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					Isolation and Characteristics of Alkaline Protease Producing Oligotrophic
Bacteria from Serpentinite Soil

Jong-Wha Lee* and Kyung-Sook Whang
Institute of Microbial Ecology and Resources, Mokwon University, Korea

   About twenty serpentine areas have located around Hongseong-Gun, Chungnam. Geochemically,
the serpentinites were not only strong alkalic ranging from pH 8.5 to 9.5, but also the concentrations of
heavy metals such as Ni, Cr, and Co ions were largely high. There were some reports on the degrees of
toxification in the serpentine areas (Kim et al., 1997).
  The present investigation was designed to evaluate the presence of oligotrophic bacteria in
serpentinite soil. Among such oligotrophic bacteria, some strains were found to be able to produce
alkaline protease. These isolates were characterized on the basis of phenotypic characteristics, cellular
fatty acid, quinone system, 16S rDNA sequence, and their taxonomic position is discussed.

Materials and Methods
• Description of the soil- Studies were carried out with soil from the adjacent metamorphic rocks and
soils from the serpentinite. Moreover, abandoned asbestos mines located around these serpentine areas,
Hong-seong and Kwang-cheon, Chungnam.
• Bacterial Counts- Bacterial numbers were determined by the dilution plate method. One ml of
adequate dilution of the soil sample with sterile distilled water was pipetted   into Horikoshi alkaline
medium(AL) ; glucose 10g, yeast extract 5g, peptone 5g, K2HPO4 1g, MgSO4 7H2O 0.2g, Na2CO3 ,
pH 10 and 100-fold dilution of AL medium(DAL).
• Determination of chemotaxonomic characteristics
- Cellular Fatty acid analysis, Quinone analysis, 16S rDNA sequencing.
• Assays of protease
- Protease activity, Effect of pH, temperature, Determination of molecular weigh.

   The samples were collected from the serpentinite soil (>pH 9.2). Moreover, abandoned asbestos
mines located around these serpentine areas, Hongseong and Kwang-cheon, Chungnam. The number
of bacteria on the diluted alkaline(DAL) medium which was diluted with 10-2 of alkaline medium(AL)
was higher than that from the full strength of Alkaline medium. Most of the isolates from diluted
alkaline plates did not show appreciable growth on full strength of alkaline medium. These isolates
were divided into four types according to the organic nutrient concentration in the growth medium
from 10-1 to 10-3 dilution of normal alkaline medium. Oligotrophic bacteria were grew in 10-3 dilution
of Alkaline medium. Among these oligotrophic isolates, sixteen alkaline protease producing bacteria
were isolated. Especially KS2D-23 and KS2D-25 strains among them were finally selected because
they appeared to produce high alkaline protease. The apparent characteristics of these isolates based on
the phenotypic, chemotaxonomic, and phylogenetic analysis are as follows. The cells are gram
negative, nonsporeforming, straight rods. Urease activity was positive. Respiratory quinone is Q-10
and major cellular fatty acids are 16:1, 16:0, 18:1, 3OH-10:0, 2OH-14:0 and 2OH-16:0. When
determining the relationship of these isolates were closely (98%) related to Variovorex paradoxus on
the basis of its 16S rDNA sequence.

Horikoshi, K. 1971. Production of alkaline enzymes by alkalophilic microorganism. Part                        . Alkaline protease
produce by Bacillus sp. No.221. Agri. Biol. Chem. 35: 1407-1414.
Kim, M. H, E. S. Min, and S. C. Song. 1997. Heavy metal contents of Gypsophila old hamiana growing on soil
derived from Serpentine. The korea J. Ecology. 20(5): 385-391.
Kobayashi, T., A. Ogasawara, S. Ito, and N. Saitoh. 1985. Purification and                     some     pro-perties of alkaline
proteinase produced by Pseudomonas maltopilia. Agri. Biol. Chem. 49: 693-698.

                                                              Comamonas testosteroni (M11224)
                                                       Xenophilus azovorans (AF285414)
                                                         Hydrogenophaga taeniospiralis (AF078768)
                                                         Variovorax paradoxus strain E4C (AF209469)
                                                         Variovorax sp. WFF52 (AB003627)
                                                         Variovorax paradoxus MBIC3839 (AB008000)
                                                  84      Variovorax paradoxus strain Iso1 (AY127900)
                                                         Variovorax sp. TUT1027 (AB098595)
                                    100                  Variovorax sp. P16G917 (AF214129)
                                                         Variovorax paradoxus (D30793)
                                            89         KS2D-25, KS2D-23
                                                          Xylophilus ampelinus (AF078758)
                                                    99         Acidovorax avenae subsp. citrulli (AF078761)
                                                              Acidovorax valerianelle (AJ431731)
                              100                                 Aquaspirillum metamorphum (AF078757)
                                                         Glacier bacterium FJI16 (AY315173)
                                                             Rhodoferax fermentans (D16211)
                                                           Rhodoferax antarcticus (AF084947)
                                                  92        Rhodoferax ferrireducens (AF435948)
                                                             Polaromonas vacuolata (U14585)
                                                  Leptothrix discophora (Z18533)
                                    100           Ideonella dechloratans (X72724)
                                                       Alcaligenes faecalis (AB091759)
                                                       Rhodospirillum rubrum (D30778)

                 Fig. Phylogenetic relationships of strains KS2D-23, KS2D-25 and
                 genera of the beta subclass of Proteobacteria base on nerar full
                 length sequence similarities of 16S rDNA.

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