Chief Editor: Satya Nugroho. Associate Editors: Bambang Sunarko, Adi Santoso, Andi Utama, Ines Atmosukarto, Wien Kusharyoto. Editorial Assistant: Siti Elly Faisholyah. Cover: Wien Kusharyoto
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Content
RAPID PURIFICATION AND PARTIAL CHARACTERIZATION OF AN EXTRACELLULAR
ENANTIOSELECTIVE LIPASE FROM ASPERGILLUS NIGER
Dominggus Malle1, Gina Garingan, Milagros Peralta
and Maria Jamela Revilleza
ABSTRACT
Lipases [EC. 3.1.1.3] are capable of hydrolyzing ester bonds of triglycerides and have received much attention from protein researchers after these enzymes have shown potential applications such us, in the manufacture of commercially important chiral drugs due to their enantioselectivity properties. A novel extracellular lipase produced by Aspergillus niger was purified and partially characterized. Using the culture medium as crude extract, the lipase was purified to 25.7 folds after Ultrafiltration and DEAE ion exchange chromatography. The enzyme was characterized to have optimum activity at neutral pH and at 30-35°C, while its kinetic parameters, Vmax and Km, were determined to be 1.80 U/mL/min and 0.2 ml, respectively. SDS-PAGE analysis revealed the presence of at least two bands (±87 and ±72 kDa), while native PAGE showed a single band. The two bands could represent subunits in a complex or isoforms of the enzyme. The use of the enzyme in the hydrolysis of a racemic mixture of 2-arylpropionic butyl ester analyzed after HPLC demonstrated an apparent predominant enantioselectivity for the S (+) enantiomer. Thus, this lipase is a promising enzyme for the chiral drug preparation of single enantiomer of 2-arylpropionic acid (ibuprofen).
Keywords: lipase, Aspergillus niger, purification, enantioselectivity, chiral drug, ibuprofen.
A HIGHLY ABUNDANT LECTIN PROTEIN IN ARABIDOPSIS THALIANA CONFERS RESISTANCE AGAINST PATHOGENS
Maria Prihtamala Omega, Skye Thomas-Hall, Peer Schenk
and Bostjan Kobe
ABSTRACT
Lectins are glycoproteins that recognize and bind to specific carbohydrates. They are involved in a range of biological functions, such as plant defence, storage proteins seed germination and plant microbe interactions. Lectin 3.1 (At3g15356) is a protein in plant model, Arabidopsis thaliana, that has been shown to be up-regulated in all defence pathways, especially in response to methyl ester jasmonate (MJ). All that was known about the gene was that it had good homology to the beta domain of legume lectins. That aim of this project was to characterize the structure and function of the lectin protein using CD spectra and X-ray crystallography. A T-DNA insertion line for the lectin gene and a number of 35S over-expression lines that had varying levels of expression had been generated, but none of these showed any obvious phenotype. Two protein bands were observed on Coomassie stained SDS-PAGE gels in the over-expression lines and in MJ induced wild-type (WT). The two protein bands represented two isoforms of the lectin 3.1 protein; in a glycosylation assay the larger protein band was shown to be heavily glycosylated. A nematode (M. incognita) disease assay discovered that the lectin over-expression lines had less nematode eggs compared to that of the WT and that the insertion line had more nematode eggs than the WT. This data provides evidence that lectin 3.1 improves plant resistance against M. incognita infection. Interestingly, the nematode gut lining contains fucose with which lectin 3.1 binds to.
Keywords: Arabidopsis thaliana, lectin’s structure and function, pathogen resistance.
ASSOCIATION BETWEEN POLYMORPHISM OF B-LACTOGLOBULIN GENE ON MILK YIELD AND QUALITY IN LOCAL SHEEP AT JONGGOL ANIMAL SCIENCE TEACHING AND RESEARCH UNIT (JASTRU)
Cece Sumantri, Nurhayati, D, Ahmad Farajallah and A. Anggraeni
ABSTRACT
The aims of this study were to identify the effects of polymorphism of â-lactoglobulin gene on milk yield and percentage of milk protein and fat in local sheep. A total number of 83 heads of lactating ewes raised under an extensive management at Jonggol Animal Science Teaching and Research Unit (JASTRU) of the Faculty of Animal Science, Bogor Agricultural University were studied. Research activities were carried out through some steps involving blood collection, DNA isolation, DNA amplification and separation of DNA fragments by electrophoresis with silver staining method. By using polymerase chain reaction (PCR) then genotyped by single strand conformation polymorphism technique (SSCP), it was successfully amplified a fragment length at 420 bp in â-lactoglobulin gene located on the exon 7. The electrophoresis pattern revealed 5 types of â-lactoglobulin gene and designated as A, B, C, D and E types. The proportion for those respective 5 types from the highest to the lowest were for A (27.71%), C (16.87%), D (12.05%), E (10.84%) and B (9.64%), respectively. There were no significant effect of polymorphisms in â-lactoglobulin gene on both milk yield and quality in local sheep at JASTRU.
Keywords: polymorphism, â-lactoglobulin gene, PCR-SSCP and local sheep.
CONSTRUCTION OF AN EPO (HUMAN-ERYTHROPOIETIN) SYNTHETIC GENE THROUGH A RECURSIVE-PCR METHOD
Asrul Muhamad Fuad, Tutus Gusdinar, Debbie Sofie Retnoningrum, and Dessy Natalia
ABSTRACT
Human erythropoietin (hEPO) is an important glycoprotein in human that is coded by a single gene named EPO (eryhtropoietin). EPO is a glycoprotein hormone that promotes erythropoiesis, which is the formation process of mature red blood cells (erythrocytes) in human bodies. It is widely used for treatment of anemia in patients with chronic renal failure. Therefore EPO has been classified as hematopoietic cytokine. Recombinant hEPO (rhEPO) has been commercially available, such as Epogen. It is produced in mammalian cells, such as CHO (Chinese hamster ovary) cells for the reason of its complex structure as a glyco-protein. In an effort to use and optimize heterologous EPO gene expression in an alternative eukaryotic host cells such as yeast, an EPO-synthetic gene (EPOsyn) was constructed. The synthetic gene had been designed to contain optimal Pichia pastoris codon usage. It had been constructed by a recursive-PCR method in two-step PCR reactions. The gene was assembled from 8 single strands synthetic oligonucleotides having an average length of 90 nt with 20 to 30 overlap region between two adjacent oligos. The synthetic gene has less GC content (45.31%) compared its native (human) gene (59.08%). The synthetic gene has been cloned in pCR2.1 cloning plasmid and sequenced. From 8 independent clones, it was revealed that the error rate was 1.59%, in which 1.42% was due to deletions and 0.17% due to substitutions. Design of the gene sequences, construction method and DNA sequence analysis of the gene will be discussed in this paper.
Keywords: Human erythropoietin (hEPO), erythropoiesis, EPO-synthetic gene, recursive-PCR, Pichia pastoris, hematopoietic cytokine.
IN VITRO PROPAGATION OF BUAH MERAH (PANDANUS CONOIDEUS LAM) THROUGH LATERAL BUD PROLIFERATION
Maria Imelda, Aida Wulansari1 and Sumarnie
ABSTRACT
Pandanus conoideus Lam or ‘Buah merah’ of the Pandanaceae is native to East Indonesia, particularly Papua and North Maluku. Traditionally the fruits are used for health promotion and maintenance as well as for curing several illnesses. Recently, it has been reported that the fruits are potential for cancer medication. As a result, there has been an overexploitation of the plants from their habitats. In order to anticipate their possible disappearance due to overexploitation in the wild, an efficient and effective technology for the mass propagation, conservation and cultivation of these plants should be developed. Generally, buah merah is propagated vegetatively by offshoots and stem cuttings or generatively by seeds. Micropropagation has many advantages over the conventional methods, because the technique allows mass clonal and pathogen-free production of plants at a high rate of multiplication all year round. In this research the effects of 0.1-0.2 mg/l thidiazuron (TDZ), 0.5-1.0 benzyl amino purine (BAP) and 0.25-0.5 mg/l Kinetin (KN) on shoot bud induction and proliferation of P. conoideus were investigated using nodal sections or lateral buds of P. conoideus on modified Murashige and Skoog medium. Shoots were rooted on MS medium without plant growth regulators (PGR). The results showed that lateral buds of Pandanus started to initiate growth after 4-7 days in culture. The best medium for shoot proliferation was MS containing either 0.5 mg/l BAP with 0.1 mg/l TDZ or 1 mg/l BAP with 0.5 mg/l KN, giving a multiplication rate of 16.5 shootlets per shootbud explant after 8 weeks. Rooting of shoots was successfully conducted on MS medium without PGR. Acclimatization of rooted plantlets was achieved on a mixed medium of cocopeat and soil (1:1).
Keywords: Buah merah (Pandanus conoideus), lateral buds, BAP, TDZ, KN.