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NOVEL MOLECULAR METHODS FOR DISCOVERY AND ENGINEERING OF BIOCATALYSTS FROM UNCULTURED MARINE MICROORGANISMS Agustinus Robert Uria; Yusro Nuri Fawzya; Ekowati Chasanah
JOURNAL OF COASTAL DEVELOPMENT Vol 8, No 2 (2005): Volume 8, Number 2, Year 2005
Publisher : JOURNAL OF COASTAL DEVELOPMENT

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Abstract

Metagenomics is a powerful cultivation-independent approach, which can be applied to gain access to the biocatalysts from uncultured marine microorganisms. Discovery of marine biocatalysts by this approach, in general, involves four main steps. First, a metagenomic library containing a pool of biocatalyst-encoding genes is constructed from a marine environment, which can be done by various methods, including cloning of enzymatically-digested DNA, uncut DNA, and PCR-amplified products. Second, the metagenomic library is screened for the genes of interest by employing the activity assay of expression product, in situ  hybridization, or Polymerase Chain Reaction (PCR). Third, the obtained target genes, both functional and phylogenetic genes, are sequenced and analysed by using bioinformatic tools in order to gain information on the functional and structural properties as well as the microbial sources of the encoded biocatalysts. Finally, the target genes are expressed in suitable microbial hosts, thereby producing the corresponding recombinant biocatalysts. All existing methods in engineering of marine biocatalysts for the performance improvement can be classified into two main strategies: (i) rational design and (ii) directed evolution. Rational design, which may include the use of resctriction enzyme(s) and splicing by overlap extension (SOE), requires information on the biocatalyst`s structural and functional properties to alter specific amino acid(s). Whereas directed evolution, including error-prone PCR technique and gene shuffling, needs no such information.
Metagenomics-Based Cloning of Amilase-Encoding Genes from the Uncultured Symbiotic Bacteria of a Marine Sponge Theonella swinhoei from Kapoposang Island, South Sulawesi Priyono, Franciscus Edi; Zilda, Dewi Seswita; Kusnadi, Yudi; Hadi, Tri A; Nurrachmi, Irvina; Uria, Agustinus Robert
Squalen, Buletin Pascapanen dan Bioteknologi Kelautan dan Perikanan Vol 12, No 1 (2017): May 2017
Publisher : Research and Development Center for Marine and Fisheries Product Processing and Biotechnol

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15578/squalen.v12i1.272

Abstract

Marine sponges have recently been recognized as the source ofenzymes, including members of hydrolases. Hydrolytic enzymes are extracellularly produced by sponge-associated bacteria to mediate the metabolism of complex organic matters, thereby assisting the sponge hosts in nutrition and metabolic processes. Among hydrolytic enzymes, amilaseshas attracted increasing attention due to their potential industrial applications. This research work was aimed atutilizing functional metagenomicsapproach for the discovery of amilases derived from the uncultured symbiotic bacteriaof the Indonesian marine sponge Theonella swinhoei. Weinitially constructed a small-insert metagenomiclibrary in Escherichia coliby cloning of metagenome in the size range of5-20 kb prepared from the sponge’s microbiome. Further functional screening of the resulting metagenomic library led to the isolation of two recombinant E. coli clones potentially harboring amilase genes, as indicated by the presence of clearing zones surroinding the selective medium containing 1% amilum. 
Identification of Protease-Producing Bacteria Isolated from Banyuwedang, Bali, and Characterization of its Protease Zilda, Dewi Seswita; Fawzya, Yusro Nuri; Uria, Agustinus Robert
Squalen, Buletin Pascapanen dan Bioteknologi Kelautan dan Perikanan Vol 13, No 3 (2018): December 2018
Publisher : Research and Development Center for Marine and Fisheries Product Processing and Biotechnol

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15578/squalen.v13i3.367

Abstract

Proteases or peptidases is known as a largest group of hydrolytic enzymes and have been applied in various industries such as food, pharmacy, leather, detergent and waste treatment. Although they are also produced by plants and animals, microbes remain the main source of proteases in the world market which mostly derived from Bacillus sp. Aims of this research were to identify isolate BII-1 and study its protease. Analysis of 16Sr RNA sequencing showed the identity of BII-1 as Bacillus subtilis (99% similarity with the same species in GenBank). It was found that protease from BII-1 exhibited optimal temperature and pH of 50 oC and 8-9, respectively. It was activated by Li2+, Na2+, Mg2+ and K+. The degenerated primer for protease gene was designed, and a partial protease gene was amplified from BII-1. The sequencing result showed that this amplified gene shared 100 and 99% similarity with those from Geobacillus thermophiles and Bacillus subtilis in the GenBank, respectively.Keywords: protease, bacteria, Bacillus subtilis, Geobacillus thermophylus
OPTIMIZATION OF Bacillus sp. K29-14 CHITINASE PRODUCTION USING MARINE CRUSTACEAN WASTE Agustinus Robert Uria; Ekowati Chasanah; Yusro Nuri Fawzya
JOURNAL OF COASTAL DEVELOPMENT Vol 8, No 3 (2005): Volume 8, Number 3, Year 2005
Publisher : JOURNAL OF COASTAL DEVELOPMENT

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Abstract

Chitin is present in large quantities in the marine crustacean waste disposed by seafood processing industries, making it very desirable as the substrate for producing chitinase, a hydrolytic enzyme of considerable interest in many industrial and agricultural applications. In our work, crustacean waste powder and its combination with colloidal chitin at different concentrations (0.5, 1.0, and 1.5%) were utilized to optimize the chitinase production by the bacterium, Bacillus sp. K29-14. The results showed that the chitinase production with the three different substrate concentrations was relatively constant in the range of 0.2 to 0.3 U/ml during 12 days cultivation, although there was a bit reduction after day 8. This activity profile seems to be similar to that of the protein content. Whereas the chitinase production on the media containing crustacean waste powder and its combination with colloidal chitin at the three concentrations showed that the highest activity (3.0 to 4.6 U/ml) was achieved on day 7 and 8. The specific chitinase activity with the waste powder at different concentrations of substrate (0.5, 1.0 and 1.5%) was increasing slowly during a nine-day cultivation. The optimal chitinase production (4.6 U/ml) was achieved with the combined substrate of 0.5% on day 8.
Teknik Peningkatan Performa Enzim Asal Mikroba Laut yang Tidak dapat dikultur Agustinus Robert Uria; Dewi Seswita Zilda; Yusro Nuri Fawzya
Squalen, Buletin Pascapanen dan Bioteknologi Kelautan dan Perikanan Vol 1, No 1 (2006): December 2006
Publisher : Research and Development Center for Marine and Fisheries Product Processing and Biotechnol

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15578/squalen.v1i1.72

Abstract

Tingginya keanekaragaman dan kelimpahan mikroba laut menunjukkan potensinya sebagai sumber paling menjanjikan untuk penemuan produk alami yang bernilai industri.  Di antara berbagai bentuk produk alami, enzim termasuk paling diminati dalam dunia industri baik dalam industri kimia, farmasi maupun makanan.  Hal ini berkenaan dengan peranannya sebagai katalis yang ramah lingkungan, ekonomis dan bersih (Wahler Reymond, 2011).
Cloning of a Transglutaminase Gene from Streptomyces thioluteus TTA 02 SDS 14 Seprianto Seprianto; Dewi Seswita Zilda; Yusro Nuri Fawzya; Suharsono Suharsono; Puspita Lisdiyanti; Agustinus Robert Uria
Squalen, Buletin Pascapanen dan Bioteknologi Kelautan dan Perikanan Vol 11, No 1 (2016): May 2016
Publisher : Research and Development Center for Marine and Fisheries Product Processing and Biotechnol

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15578/squalen.v11i1.189

Abstract

Microbial Transglutaminase (MTGase, EC 2.3.2.13) is an enzyme that catalyzes the transfer of acyl group. Many microbial strains that produce MTGase belong to Streptomyces members. This research was aimed at cloning of a MTGase gene. PCR–based screening of ten MTGase-producing streptomyces isolates from soil in West Nusa Tenggara led to detection of one potential isolate, designated as TTA 02 SDS 14. The partial  MTGase-encoding gene (470 bp)  was amplified by PCR and sequenced. The sequence result indicate its similarity of 93 % with that of Streptomyces cinnamoneus. The 16S rRNA gene analysis showed its identity as Streptomyces thioleteus. Fosmid-based construction of a genomic library from the isolate  and subsequent screening led to the isolation of  a ~40-Kb fosmid harboring a MTGase gene.