31 CLONING OF A TRANSGLUTAMINASE GENE FROM Streptomyces thioluteus TTA 02 SDS 14 Seprianto 1 , Dewi Seswita Zilda 2* , Yusro Nuri Fawzya 2 , Suharsono 1 , Puspita Lisdiyanti 3 and Agustinus Robert Uria 2* 1 Departement of Biotechnology, Bogor Agricultural University, Indonesia, Jalan Raya Dramaga Kampus IPB, Bogor 16680, Indonesia 2 Research and Development Center for Marine and Fisheries Product Competitiveness and Biotechnology, Jalan KS Tubun Petamburan VI, Central Jakarta 10260; 3 The Research Center for Biotechnology, Indonesian Institute of Sciences Abstract Microbial Transglutaminase (MTGase, EC 2.3.2.13) is an enzyme that catalyzes the transfer of acyl group. It has tremendous applications in binding meats together in texture improvement process. This research was aimed at cloning MTGase gene from Streptomyces thioluteus TTA 02 SDS 14. A genomic library with the average of 40 kb insert size was initially generated from the total genomic DNA of S.thioluteus TTA 02 SDS 14, and was subsequently screened using an MTGase-targeting primer pair. The genomic library construction was based on the fosmid pCC1FOS TM hosted in E.coli EPI300-TI R . The isolated positive clone was verified by enzymatic digestion with NotI, EcoRV, and BamHI, followed by PCR-amplification of MTGase fragment. The PCR product result of 360 bp was sequenced. The sequence analysis strongly suggests that the positive clone with 40-kb insert harbors an MTGase gene. Keywords: MTGase gene, S. thioluteus TTA 02 SDS 14 , genom library Article history: Received: 15 January 2016; Revised: 8 April 2016; Accepted: 28 April 2016 Squalen Bull. of Mar. and Fish. Postharvest and Biotech. 11 (1) 2016, 31-36 www.bbp4b.litbang.kkp.go.id/squalen-bulletin Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology ISSN: 2089-5690 e-ISSN: 2406-9272 *Corresponding author. E-mail: seswitazilda@yahoo.com; agustinus.uria@gmail.com Copyright © 2016, Squalen BMFPB. Accreditation Number: 631/AU2/P2MI-LIPI/03/2015. DOI: http://dx.doi.org/10.15578/squalen.v11i1.189 1. Introducton Transglutaminase (TGase, E.C. 2.3.2.13) is an enzyme that catalyzes the transfer of acyl group. Indirectly, the a-carboxyamide groups of peptide-bond glutamine residues (Gln) act as the acyl donors, and the ε-amino groups of lysine residues (Lys) serve as acyl acceptors, intermolecular or intramolecular ε-(γ- glutamyl) lysine bonds that are formed, resulting in the polymerization of food proteins (Griffin et al., 2002). TGase has been used for texture improvement (e.g. gelling capacity, hardness and elasticity) of food protein-based products and fishery product processing, such as surimi or fish meat-based processed products. Indonesian product processing has grown rapidly in line with the increase of fish production and the development of fish products processing. TGase is widely spread in nature, including in animals, plants, and microorganisms. In animals, it is involved in various biological functions, ranging from blood clotting to cell differentiation (Aeshclimann & Paulsson, 1994). This enzyme’s limited sources and complicated separation and purification process make it very expensive (around US$ 80/unit) (Zhu et al.,1995). TGases of microbial origin (MTGases) have recently received increasing attention for industrial applications. The properties of MTGases offer more advantages compared to animal-derived transglutaminases, such as no requirement of cofactor Ca 2+ for their activity, their stability across wide pH range, their ability to cross-link most food proteins, and lower production costs due to easy enzyme production and purification (Langston et al., 2006). Therefore, TGase production based on microorganisms is worthy to be pursued. However, using wild-type microorganisms to product MTGase involves several drawbacks, such as slow cell growth, low yield, a large amount of non- target proteins interference to the target proteins, and the difficulty to purify native proteins that requires multiple purification steps (Soares et al., 2003). DNA recombinant technology is an emerging and well- established approach to provide bacterial enzymes in