01 Cahyani.cdr Vol.11, No.1, March 2017, p 1-10 DOI: 10.5454/mi.11.1.1 Cloning of Synthetic Lipase Gene from Rhizomucor miehei with Original Signal Peptide in Pichia pastoris 1 2 2 1 MARTHA EKA CAHYANI , IS HELIANTI *, NIKNIK NURHAYATI , AND ABINAWANTO 1 Department of Biology, Faculty of Mathematics and Natural Science, Universitas Indonesia, Depok 16424, Indonesia; 2 Center of Bioindustrial Technology, Agency for Assessment and Application of Technology (BPPT), Building 611, LAPTIAB-BPPT, Puspiptek Area, Tangerang Selatan 15314, Indonesia Lipases (EC 3.1.1.3) are classified as hydrolases that hydrolyze lipids. These enzymes have potential application in biotechnology and industrial process. In previous study we have cloned the synthetic Rhizomucor miehei lipase gene using the vector pUC57 in Escherichia coli DH5α, but only found the very low enzymes activity. This study aimed to clone synthetic Rhizomucor miehei lipase gene into Pichia pastoris expression plasmid for lipase expression with the original signal peptide. A DNA fragment with the original signal peptide had been obtained by PCR, cut by Xho I and Xba I and then ligated into pPICZα A linearized with the same enzymes. The mixture of ligation reaction, then was transformed into Escherichia coli DH5α. Zeocin-resistant transformants were selected and contained plasmid was analyzed by restriction enzymes analyses and DNA sequencing. As the result, a synthetic Rhizomucor miehei lipase gene (RMlip) with the size of 1132 bp was successfully cloned to pPICZα A vector plasmid. The recombinant plasmid with the correct DNA sequence was transformed into Pichia pastoris X33. Cultivation of recombinant P. pastoris was carried out with the addition of 1.5% methanol every day with appropriate aeration. The recombinant lipase produced by Pichia pastoris X33 o containing RMlip in its chromosomal DNA had optimal temperature and pH, 30 C and 9.0, respectively. Key words: Pichia pastoris, pPICZα A, Rhizomucor miehei, synthetic lipase gene Lipase (EC 3.1.1.3) diklasifikasikan sebagai enzim hidrolase yang menghidrolisis lipid. Enzim ini memiliki potensi dalam berbagai aplikasi di bidang bioteknologi dan industri. Dalam penelitian sebelumnya, kami telah mengklon gen lipase Rhizomucor miehei sintetik menggunakan vektor pUC57 dalam Escherichia coli DH5α, dan mendapatkan aktivitas enzim yang sangat rendah. Penelitian ini bertujuan untuk mengklon gen lipase Rhizomucor miehei sintetik ke dalam plasmid ekspresi Pichia pastoris untuk mengekspresikan lipase dengan menggunakan peptida sinyal asli. Gen lipase sisipan diperoleh dengan PCR, dipotong dengan Xho I dan Xba I, kemudian diligasi ke dalam pPICZα A yang telah dilinearisasi dengan enzim yang sama. Hasil ligasi kemudian ditransformasi ke dalam Escherichia coli DH5α. Transforman yang tahan terhadap zeocin dipilih dan kandungan plasmidnya dianalisis dengan enzim restriksi dan pengurutan DNA. Hasilnya diperoleh gen lipase Rhizomucor miehei (RMlip)berukuran 1132 pb. Plasmid rekombinan yang tepat kemudian ditransformasikan ke Pichia pastoris X33. Kultivasi dilakukan dengan penambahan 1.5% metanol setiap hari dengan aerasi yang sesuai. Lipase rekombinan yang diproduksi oleh Pichia pastoris X33 yang mengandung RMlip di dalam DNA o kromosomnya mempunyai temperatur dan pH optimal masing-masing 30 C dan 9. Kata kunci: gen lipase sintetik, Pichia pastoris, pPICZα A, Rhizomucor miehei MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: +62-21-7560536 ext 7119, Fax:+62-21-7566922; Email: is.helianti@bppt.go.id Vandenberghe 2003). Lipase used commercially mainly come from microorganisms. Microbial lipases chosen because it has a more stable properties such as selectivity and broad substrate specificity (Dutra et al. 2008; Griebeler et al. 2011). One of the microorganisms produces lipase is Rhizomucor miehei (Sarma et al. 2001). R. miehei is a mold that has lipase with high activity and good stability under diverse conditions (Rodrigues and Lafuente 2010). Lipase of R. miehei has been used in in ester hydrolysis, ester synthesis, and transesterification reaction (Huang et al. 2012). These lipase was able to successfully catalyze the synthesis of ethyl caproate in organic solvent displayed on the surface Pichia pastoris (Han et al. 2009), and also effective for the Lipase (EC 3.1.1.3) is classified as hydrolase that hydrolyzes lipids. These enzymes are biocatalysts that can act on solid and liquid media (Brunel et al. 2004). This character makes lipase potential in a variety of applications in biotechnology and industry, including pharmaceutical, pesticide, biosensors, biodiesel, detergents, and various food products (Vakhlu and Kour 2006). Several techniques have been developed to obtain higher conversions, as highly specific enzymes for each application, improving the possibility of industrial applications for these biocatalysts (Franken et al. 2008; Soccol and transesterification of vegetable oils and other raw materials containing higher fatty acid alkyl ester into derivative (Huang et al. 2014). Since then, industry has continually searched for new components to improve the benefit of this development along with its cost- efficiency. Center of Bioindustrial Technology, LAPTIAB, BPPT has conducted cloning of synthetic Rhizomucor miehei lipase gene using the vector pUC57 in Escherichia coli DH5α. Although the cloning process succeeded, but the enzyme activity obtained is very low. In this study we chose Pichia pastoris as a host to replace E. coli. P. pastoris is a methylotrophic yeast that has been considered as an excellent host for the production of heterologous proteins (Sreekrishna et al. 1997). P. pastoris has distinguished characteristics, yielding stable transformants of integration of foreign DNA into chromosomal DNA, have a high-density growth, have the ability to secrete high levels of foreign proteins (Adrio and Demain 2003), expressing heterologous protein can either be intracellular and extracellular (Balamurugan et al. 2007). In addition, they have the simplicity of techniques needed for the molecular genetic manipulation and the ability to modify the eukaryotic protein after translation, such as glycosylation, disulfide bond formation and proteolytic processes (Cereghino and Cregg 2000). Expression of any foreign gene in P. pastoris requires three basic steps, which is the insertion of the gene into an expression vector, introduction of the expression vectors into the genome of P. pastoris, and examination of potential expression strains for the foreign gene product (Cereghino et al. 2000). Therefore, this study aimed to clone R. miehei synthetic lipase gene in P. pastoris expression plasmid for lipase expression with the original signal peptide. MATERIALS AND METHODS Strains, Plasmids, and Media. E. coli DH5α (Invitrogen) was used as the host for the recombinant plasmid cloning and propagation. P. pastoris X33 was used as the host for lipase expression. Plasmid pUC57 containing the DNA of synthetic R. miehei lipase (RMlip) has been described elsewhere (Hanniya 2015). The vector pPICZα A was supplied from o Invitrogen. E. coli DH5α was grown at 37 C in Luria Bertani (LB) low salt agar medium containing 25 μg -1 mL zeocin for selection of clones transformed with the recombinant vector plasmid. P. pastoris X33 was o routinely grown in shaking flasks at 30 C, in a rich standard medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate - buffer, pH 6.0, 1.34% (w/v) yeast nitrogen base, 4 x 10 5 % (w/v) biotin, 1% (v/v) glycerol (Buffered Glycerol- complex (BMGY)) before induction, or 1.5% (v/v) methanol (Buffered Methanol-complex (BMMY)) for induction. For maintaining cultures and plates, Yeast Extract Peptone Dextrose (YPD) medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose) -1 containing 50 μg mL zeocin was used, and for selection of transformants, Yeast Extract Peptone Dextrose Sorbitol (YPDS) medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose, 1 M -1 sorbitol, 2% (w/v) agar) containing 50 μg ml zeocin and Tributyrin Agar (TBA) medium (1% (w/v) yeast extract, 2% (w/v) special peptone, 2% (w/v) tributyrin, 2% (w/v) agar, 2% (w/v) dextrose) containing 50 μg -1 mL zeocin was used. Construction of the Vector pPICZα A-RMlip. Construction of the vector pPICZα A-RMlip including cloning, transformation into E. coli and selection, were conducted as described by Sambrook and Russel (2001). The E. coli expression vector pUC57 containing the synthetic RMlip gene served as a template for polymerase chain reaction (PCR). PCR w a s p e r f o r m e d u s i n g f o r w a r d p r i m e r ( 5 ' - GCATCCTCGAGAAAAGAGAGGCTGAAGCTAT GGTGCTGAAACAGCGCGC-3') and reverse primer (5'-GCATCTCTAGAGCGGTGCACAGGCCGGTG TTAATG-3'). The forward primer containing an Xho I- site (underlined) and the reverse primer containing an Xba I-site (underlined) and the sequence that will detect 6xHis. The primers were ordered from Integrated DNA Technologies (Singapore). The PCR process is carried out by 30 cycles with predenaturation, denaturation, o o annealing, extantion, endextantion at 98 C, 30 s; 98 C, o o o 10 s; 69 C, 30 s; 72 C, 30 s; 72 C, 5 min, respectively. The obtained insert gene was digested with Xho I and Xba I. The restriction enzymes were purchased from Fermentas (Burlington, Canada) and Thermo Scientific (Waltham, USA), respectively. It was then ligated into pPICZα A linearized with the same enzymes. The mixture of ligation reaction, then was transformed into E. coli DH5α. Zeocin-resistant transformants which grown on LB low salt media were selected and analyzed by plasmid digestion with Bgl II, Xho I, and Xba I. Transformants showed the correct digestion pattern were sequenced to confirm the sequence. Transformation into P. pastoris. Plasmid with the correct DNA sequence, were used for transformation into 2 CAHYANI ET AL. Microbiol Indones Volume 11, 2017 Microbiol Indones 3 P. pastoris X33. The yeast was transformed with 1 μg of Sac I-linearized pPICZα A-RMlip by the spheroplast method of Cregg et al. (1985) with the following modifications. A fresh colony of P. pastoris X33 was inoculated into 10 mL of YPD and grown with shaking at o 250 rpm 30 C overnight. On the following day, 5 mL sample of the overnite culture was added to 45 mL of fresh YPD as starter OD of 0.2 and grown cells under 600 the previous conditions until OD reaches 1.4 to 1.6. The 600 cells were harvested by centrifugation at 6000 x g for 5 min and washed twice with 10 mL of ice-cold miliQ H O 2 and then suspended in 8 mL of freshly prepared T buffer (10 mM Tris-HCl pH 7.5, 0.1 M Lithium acetate, 10 mM DTT, 0.6 M sorbitol) and incubated at 30 °C for 30 min. The spheroplasts were washed twice in 10 mL of ice-cold 1 M sorbitol and suspended in 400 μL of ice-cold 1 M sorbitol. One hundred μL of the spheroplasts were mixed with 1 μg of Sac I-linearized pPICZα A-RMlip. Then, the transformation performed by electroporation, which is applied electric pulse to the mixture (1.5 kV, 25 μF, 200 Ω for Gene pulser, Bio-Rad, USA) The mixture was added o by 1 mL of ice-cold 1 M sorbitol and incubated at 30 C without shaking for 1 h. The mixture then was added by o 1 mL of YPD and incubated at 30 C with shaking at 250 rpm for 1 h. One hundred μL of the mixture were -1 spreaded on TBA medium plates containing 50 μg mL o zeocin and incubated at 30 C. Transformants were o selected after 2-3 days at 30 C by plating cells on TBA medium. Lipase activity was detected by the appearance of clear zones around colonies. Zeocin-resistant transformants which showed a clear zone were selected and analyzed by colony PCR and continued to PCR product digestion. Cultivation. Standard cultivation method was carried out according to method described in Hu et al. (2013) and Wang et al. (2016). A single positive colony o was cultivated in 100 mL of BMGY medium at 30 C overnite under constant agitation at 250 rpm, until an OD = 2-5 was reached. Then, these culture was 600 transfered into 900 mL of the BMGY medium for large- scale cultivation until an OD reached 5. The cells 600 were collected by centrifugation at 3800 rpm for 20 min, and resuspended in 200 mL of the BMMY medium. Cultures were induced with absolute methanol to a final concentration of 1.5% every 24 h. The expression culture supernatants was harvested o every 24 h and stored at -4 C before analysis. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The expression of lipase gene in the supernatant was analyzed by SDS- PAGE, which was conducted using a 12% polyacrylamide gel on a vertical mini gel apparatus (40 A, 200 V, 45 min, Bio-Rad, USA), as described by Laemmli (1970). Protein molecular weight marker was purchased from Amersham (GE Healthcare, UK). Samples were mixed with 2× loading buffer, with a ratio 1:1. Then, the mixture was boiled at 100 °C for 5 min before electrophoresis. Proteins were stained with page blue staining solution (Thermo Scientific, Waltham, USA). Protein staining is done by polyacrylamide gel washing with miliQ H O three 2 times, then incubated with page blue for 30 s in the o microwave and continued incubation at 24 C, 300 rpm, for 1 h. Furthermore, it was rinsed and soaked by miliQ H O overnight.2 Characterization of Recombinant Lipase. The lipase properties of supernatants were determined by the alkali titration method, using olive oil as substrate, as described by Fang et al. (2014) with the following modifications. The reaction was conducted in a mixture of 4 mL of 50 mM Tris–HCl (pH 8.0), 5 mL of emulsion of olive oil (25% (v/v) olive oil emulsified with 1.5% (w/v) polyvinyl alcohol solution) and 1 mL of proper o dilute enzyme solution at incubation 50 C for 20 min in a shaker. Finally, 5 mL of methanol was added to terminate the reaction. The amount of liberated fatty acids was measured by titration with 50 mM NaOH using phenolphthalein as an indicator. One unit (U) of lipase activity was defined as the amount of lipase necessary to liberate 1 μmol per min of fatty acids from the olive oil. The optimal tempetarure was determined by examining the activity of the enzymes at Tris-HCl buffer pH 8.0 in the various temperatures incubation o (30-80 C). The optimal pH was determined by measuring the enzymatic activity at the optimal temperature at pH range 5.0-10.0 (pH 5.0; pH 6.3-8.0; pH 8.0-9.0; pH 9.4-10.0 using Na-citrate buffer 0.05 mM; Na-phosphate 0.05 mM; Tris-HCl 0.05 mM; Glycine-NaOH 0.05 mM, respectively). Each sample was assayed in duplicate and the average value was determined. RESULTS Construction of the Recombinant Plasmid pPICZα A-RMlip. A fragment DNA encoding synthetic R. miehei lipase gene (RMlip) with the total size 1132 bp has been obtained by PCR. The vector pPICZα A is a P. pastoris expression vector. These vector size of 3593 bp. In order to the vector and RMlip can be ligated, the vector was also restricted at Xho I and Xba I sites. The total size pPICZα A-RMlip after Fig 1 E. coli transformants verification. (A) Analysis of transformants by plasmid digestion with Bgl II. (B) Analysis of transformants by plasmid digestion with Xho I. Line 2: Negative control. Lines 3-8: Transformants analyzed with enzime restriction, the sizes of 2992 bp and 1629 bp or 4629 bp, respectively. (C) Analysis of transformants by plasmid digestion with Xba I. Lines 2-7: Transformant analyzed with Xba I, the sizes of 4629 bp. Line 2: Negative control. Each transformant was screened in duplicate. ligated is 4621 bp. This ligated vector and inserted fragment was then transformed into E.coli. After transformation, there were four transformants grown on LB low salt agar medium. Of the four transformants, only three transformants can be grown in LB low salt broth medium. Three transformants cultures were verified by Bgl II, Xho I, and Xba I. Xho I and Xba I are the single cutting site located between the vector pPICZα A and RMlip, so that the DNA size obtained by cutting was 4621 bp (Fig 1A and 1B). While, Bgl II is the double cutting sites located at the vector pPICZα A and RMlip, so that the result was obtained by cutting the size of 2992 bp and 1629 bp (Fig 1A). Based on the verification by Bgl II, Xho I, and Xba I, showed that the three transformants are three positive colonies that carrying the vector pPICZα A-RMlip with a total size of 4621 bp. Sequencing. Sequence analysis was conducted to confirm the accuracy of the target gene sequence, the correct fusion of lipase gene to the vector, and the rate of mutation that occurred during the selection process. From DNA sequencing result we found that the DNA was in frame with alpha factor. The sequences were processed using online software www.genome.jp/ tools/clustalw/, later translated using software http://web.expasy.org/translate/ into the protein sequence of samples. Based on the results of the sequencing, there are primers used (grey highlight), which is used as a marker of the original signal peptide, a marker of restriction enzymes Xho I and Xba I, as well as the marker His codon before the end translation process (Fig 2A). Sequence of synthetic R. miehei lipase is between the both primers. Based on the protein sequence, there are amino acid of the original signal A B C 4 CAHYANI ET AL. Microbiol Indones Volume 11, 2017 Microbiol Indones 5 peptide, amino acid of the propeptides, and amino acid of the mature enzyme, and obtained also size lipase protein produced is 32.9 kDa (Fig 2B). The results of the translation are then alignment with international protein sequence databases available on the website http://ncbi.nlm.nih.gov by selecting Protein BLAST menu. Alignment is performed to find the sample sequence similarity to sequences contained in the databases. Protein BLAST results showed that the protein sequence pPICZαA-RMlip transformed into E. coli DH5α have similarities that are 100% identical to the protein lipase from Rhizomucor miehei in international database (data not shown). Transformation into P. pastoris X33 and Selection of Positive Transformants. After transformation process of the correct sequence recombinant plasmid into P. pastoris, the recombinant colonies were grown. Transformants expressing lipase activity, as indicated by the formation of a clear zone around colonies on TBA medium. Several transformants were picked for confirmation by colony PCR and continued plasmid digestion with Bgl II. The total size of RMlip after the colony PCR was 1132 bp and the total size of RMlip after the restriction by Bgl II was 680 bp and 452 bp. Based on the confirmation by the colony PCR and Bgl II, showed that the transformants are positive colonies that carry the vector pPICZα A-RMlip (Fig 3). SDS-PAGE Electrophoresis. SDS-PAGE analysis showed that after induction by methanol, there was a band that appeared after induction with the measured molecular weight 32.9 kDa (Fig 4). Characterization of Lipase Properties. Temperature and pH are two important reaction parameters for enzymes. Each enzyme has a distinct optimal temperature and pH levels. As well as recombinant lipase produced from recombinant P. pastoris X33 containing RMlip. The optimal reaction temperature and pH of this recombinant lipase o were 30 C and 9.0, respectively. DISCUSSION Lipases has been successfully applied in various applications, such as in the food industry, organic synthesis, pharmaceutical (Houde et al. 2004; Diego et al. 2009) and in the field of biodiesel (Huang et al. 2012; Huang et al. 2014). The success is challenging us to keep looking for a better lipase expression with the aid of genetic engineering. This study aimed to clone R. miehei synthetic lipase gene into P. pastoris expression plasmid for lipase expression with the original signal peptide. In this study R. miehei synthetic lipase gene with signal peptide has been cloned in P. pastoris expression vector and transformed into P. pastoris X33. Based on the analysis sequensing, RMlip has 363 amino acid residues, which consists of a 24 amino acid residues signal peptide, a 80 amino acids residues propeptida, and 269 amino acid residues of the mature enzyme. This is supported by Boel et al. (1988), which said that, RMlip is synthesized as a precursor, which contained 70 amino acid residues propeptida before the 269 amino acid residues mature enzyme. Differences in the number of amino acids propeptides affected by signal peptides are used. That is because propeptides are C-terminal domain of signal peptides containing site of the cutting, which will be cleaved off in the protein maturation steps (Emanuelsson et al. 2007; Wang et al. 2016). Signal peptides and propeptides is a very important sequence in protein synthesis, because the functional region of the protein cannot form the native structure without the propeptide region (Anderson et al. 1999). In addition, the protein molecular weight of lipase in pPICZα A-RMlip is 32.9 kDa. This is reinforced by the statement Wu et al. (1996) which says that the molecular weight of RMlip is 31.6 kDa and the statement Huge-Jensen et al. (1989) states that, the molecular weight of RMlip is 32 kDa. So it is suspected that the molecular weight of lipase RMlip is in the same range. Temperature and pH is a very important parameter to determine the properties of the lipase expressed (Wang et al. 2015). In this study, RMlip express lipase -1 with high levels at 30 °C, ie at 8.33 U mL ± 0.50. This is supported by the statement Huang et al. (2014), having said that, the temperature tolerance for RMlip well expressed in P. pastoris X33 is at a temperature of o 0-40 C. For optimum pH, RMlip express lipase with high levels at pH 9.0 in Tris-HCl buffer, ie at 8.44 U -1 mL ± 0.07. This is supported also by the statement Huang et al. (2014) which says that, pH tolerant for RMlip in P. pastoris X.33 ie at pH 4.0—10.0. He et al. (2015) has been conducting research to increase the activity of lipase RMlip by combining the AOX1 and GAP promoter whose expression using the α factor signal peptide of the Saccharomyces cerevisiae in P. pastoris GS115, lipase activity is generated is 140 U -1 mL . The result is higher of the control that only used -1 the AOX1 and GAP promoter, which is 25 U mL and -1 20 U mL , respectively. When compared with the control and the results of the such research, the lipase Fig 2 Analysis sequencing. (A) In the DNA sequence, there are primers used (grey highlight), which is used as a marker of the original signal peptide, a marker of restriction enzymes Xho I and Xba I, as well as the marker His codon before the end translation process, sequence of synthetic Rhizomucor miehei lipase is between the both primers. (B) In the protein sequence, there are amino acid of the original signal peptide, amino acid of the propeptides, and amino acid of the mature enzyme. These components are very important in protein expression. The protein sequence analysis by software http://web.expasy.org/ translate/ showed that the size of pPICZαA-RMlip lipase protein molecular weight is 32.9 kDa. Theoretical pI/Mw: 5.03 / 32917.74 A B Region of Rhizomicor miehei lipase Part of the a factor of pPICZaA Region of signal peptide and propeptides 6 CAHYANI ET AL. Microbiol Indones activity generated in this study has not increased significantly. It may caused by several factors, the most striking is the incorporation of the promoter and signal peptide used. The combined use of the AOX1 and GAP promoters can be attributed to a positive effect on the expression level by increasing the copy number of the expression cassette. The transcription levels of the intracellular protein and secreted protein have been shown to increase greatly upon integration of multiple copies of the expression vector to an appropriate extent in P. pastoris (Scorer et al. 1994; Shen et al. 2012). Signal peptides also affect the expression of recombinant lipase. There are three kinds of signal peptides that can be used directly in Pichia pastoris to secrete and express a foreign gene that is the native signal peptides, the S. cerevisiae α factor prepropeptides (α factor signal peptide) and the P. pastoris acid phosphatase (PHO1) signal. The original/native signal peptide is rarely used because there are still some weaknesses, one of which is described in the research Brocca et al. (1998) regarding the effect of the original signal peptides on the expression and secretion of industrial lipase Lip1 from Candida rugosa. Brocca et al. (1998) stated that the A B Fig 3 Pichia pastoris transformants confirmation. (A) RMlip confirmation result of transformation by colony PCR. Line 1: GeneRuler DNA Ladder 1 Kb. Line 2: Negative control of PCR that used ddH O. Line 3: 2 Positive control of PCR that used extraction RMlip. Line 4-8: RMlip result of transformation, the total size was 1132 bp. Almost all the positive colonies, from five colonies, only 1 negative colony. (B) RMlip confirmation result of colony PCR by Bgl II. Line 1: GeneRuler DNA Ladder 1 Kb. Line 2: Negative control of PCR that used ddH O. Line 3: Positive control of PCR that used extraction RMlip. Line 4—7: 2 RMlip result of transformation, the total size was 680 bp and 452 bp. Restriction results from four colonies, are all positive. Fig 4 SDS-PAGE analysis of culture supernatans during the cultivation. The molecular weight of protein RMlip is 32.9 kDa. Line M: Protein molecular weight marker. Line 1-6: Culture supernatant after 0, 24, 48, 72, 96, 120, and 144 h of cultivation. Volume 11, 2017 Microbiol Indones 7 original signal peptide can secrete lipase Lip 1, but its expression was hampered. There is an assumption that, in this study also experienced the same thing, signal peptide RMlip successfully constructed, but the expression of lipase inhibited. Cereghino and Cregg (2000) said that, although the original signal peptide and α factor prepropeptida (α factor signal peptide) from S. cerevisiae was adequate for foreign protein secretion and expression, but the highest levels of protein secretion was from clone with the full preprotein. This study obtained that synthetic Rhizomucor miehei lipase gene with the size of 1132 bp was successfully cloned to pPICZα A vector with a total size of 4621 bp. DNA sequencing analyses showed that the synthetic lipase gene pPICZα A-RMlip are 100% identical to the R. miehei lipase. The gene pPICZα A- RMlip was successfully integrated into chromosome of P. pastoris X33. The enzymatic properties of pPICZαA-RMlip in P. pastoris X33 is to have the o optimal reaction temperature is 30 C and the optimal reaction pH is 9.0. ACKNOWLEDGMENT This study was funded by National Insentive for Consortium Research (Insinas) 2015—2016 from Ministry of Research, Technology, and Higher Education, Republic Indonesia. REFERENCES Adrio JL, Demain AL. 2003. Fungal Biotechnology. Int. Microbiol. 6: 191-199. doi: 10.1007/s10123-003-0133-0. Anderson DE, Peters RJ, Wilk B, Agard DA. 1999. Alphalytic protease precursor: characterization of a structured folding intermediate. 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