S. Gaffar et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 273-279) Jatinangor, 30-31 October 2008 273 Signal peptide modification of α-amylase Gene (ALPI) and construction of pPICZA-MSALPI plasmid for improving secretory production of α-amilase in Pichia pastoris Shabarni Gaffar 1 *, Dessy Natalia 2 , Maelita R. Moeis 3 , O. Suprijana 1 , Soetijoso Soemitro 1 1 Department of Chemistry, FMIPA, Universitas Padjadjaran 2 Department of Chemistry, FMIPA, Institut Teknologi Bandung 3 School of Natural Science Technology, Institut Teknologi Bandung *email: sabarni.ghafar@unpad.ac.id Abstract α-Amylases hydrolyze the α-1,4-glycosidic bond in starch results in maltose and oligosacharides. The enzyme has been widely used in various industries, such as starch liquefaction, textile, brewing, baking, detergent, and paper industries. To improve secretory production of S. fibuligera -amylase in Pichia pastoris, in this research the signal peptide of S. fibuligera α-amylase was genetically engineered with modified signal peptide (MS). MS was designed to contain the pro region of the signal peptide of Saccharomyces cerevisieae α-mating factor fused to 15 residue amino acid of mouse salivary α-amylase signal peptide to replace the wild type signal peptide of α-amylase. Modification of signal peptide was done using a set of primers 5’MSPαF and 3’ALPIApa by PCR (Polymerase Chain Reaction) method and pPICZαA-ALPI as the template. The resulted 1,7 kb DNA fragment of MSALP1 was subcloned into pGemT plasmid and subsequently into pPICZA expression vector. Restriction and DNA sequence analysis showed that signal peptide of S. fibuligera α-amylase has been successfully modified and pPICZA-MSALPI has been successfully constructed. Qualitative analysis of α-amylase shown that P. pastoris transformants which contain recombinant α-amylase, secreted α-amylase into growth medium indicated by clear zone on YPD medium containing 1% starch-KI/I 2 . Key words: α-amylase, signal peptide, P. pastoris, pPICZA-MSALPI Introduction Saccharomycopsis fibuligera is a food-borne, dimorphous yeast, which has been considered as one of the best producers of amylolytic enzymes (Hostinova, 2002). S. fibuligera R-64 is known as a strain that produces α-amylase (1,4-α-D-glucan-4- glucanohydrolase, E.C.3.2.1.1) which has been screened from 136 isolates in Indonesia (Soemito et.al., 1996). This enzyme catalyses the hydrolysis of α-1,4-glycosidic linkages of raw and soluble starch, thereby generating glucose, maltose, smaller dextrins and oligosaccharides (Sanoja et.al., 2005). α-amylase has been widely used in food industry, beverage, pharmacy, textile and detergent (Cowan, 1996; Hashida et.al. 2000). Indonesia is still importing this enzyme because of the lack of domination of technology to produce industrial scale enzyme. Hasan et.al. (2007) was reported that S. fibuligera R-64 α- amylase has been identified as a raw starch degrading enzyme, which is advantageous for industrial application. Unfortunately, S. fibuligera R-64 produces a low α-amylase secretion level. One of constraint to produce wild type α- amylase is the low of its productivity, while industry requires microbes with economic and high productivity. To yield microbe strain with this pre- eminent characters, recombinant DNA technology can be exploited. The enzymes can be yielded in heterologous protein expression system, such as E. coli, S. cerevisiae and P. pastoris. Number of enzymes secreted from wild type microbe is usually lower compared to recombinant enzymes (Hashida et. al. 2000; Glick & Pasternak, 2003). During the past 15 years, the methylotrophic yeast Pichia pastoris has been developed into highly successful system for production of a variety of the heterologous protein. The increasing popularity of this particular system can be attributed most several factors, most importantly: (1) the simplicity of techniques needed for molecular genetic manipulation of P. pastoris and similarity to those of Saccharomyces cerevisiae, one of the most well- characterized experimental system in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, intracellular and extra cellular; (3) the capability of performing many eukaryotic post-translational, such as glycosylation, disulfide bond formation, proteolysis processing, and (4) the availability of expression system as a commercially available kit (Cereghino & Cregg, 2000). Potential bottlenecks for protein secretion include (1) the codon usage of the expressed gene developed as widely used host organism for ISBN 978-979-18962-0-7