Glukosa Oksidase Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene.

Presentasi berjudul: "Glukosa Oksidase Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene."— Transcript presentasi:

1 Glukosa Oksidase Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene Ji-Hyun Ko, Moon Sun Hahm,a Hyun Ah Kang, Soo Wan Nam and Bong Hyun Chunga Protein Expression and Purification 25 (2002) 488–493 Syahfitri Anita

2 Agenda Properti Glukosa Oksidase Aplikasi Glucose oksidase
Latar Belakang Tujuan Penelitian Metode Hasil Kesimpulan

3 Glukosa Oksidase (GOx)
EC FAD sebagai kofaktor The reaction can be divided into, a reductive and an oxidative step (Scheme 1.1). In the reductive half reaction, GOX catalyses the oxidation of β-D-glucose to Dglucono-δ-lactone which is non-enzymatically hydrolyzed to gluconic acid. Subsequently the flavine adenine dinucucleotide (FAD) ring of GOX is reduced to FADH2 (Witt et al., 2000). In the oxidative half reaction the reduced GOX is reoxidised by oxygen to yield hydrogen peroxide.

4 Mekanisme +O2 -H2O2 +D-Glucose -D-Gluconolactone

5 Properti GOx Terdapat di serangga (lebah, belalang) dan jamur (Aspergillus niger, Penicillium sp dan Botrytis cinerea). Berat Molekul bervariasi antara 150kDa-186kDa (Tsuge et al ) pH optimum 4–7 (Keilin and Hartree 1947; Nakamura and Fujiki 1968; Bao et al. 2001) Titik Isoelektrik (pI) 4.2 (Pazur and Kleppe 1964) Nilai Km GOX is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent. Honey preservation pada lebah, anti-mikroba (hardening cuticle) pada belalang, antibakteri dan antifungal pada penicillum sp. Km (mM) pH T (°C) Referensi 198–248 5–7 20–30 Bao et al. 2001 110–120 5.6 0–38 Gibson et al. 1964 50–74 5.5 15–30 Nakamura and Ogura 1968b 33 25 Swoboda and Massey 1965

6 Struktur Struktur 3 dimensi glukosa oksidase dari Apergillus niger
below. Each GOX subunit contains one mole of tightly bound, but not covalently linked, flavine adenine dinucleotide (FAD) as co-factor (Rando et al., 1997 and Witt et al., 2000). The GOX enzyme from P. amagasakiense is glycosylated, with a carbohydrate content of approximately 11-13%, which is of the high mannose type

7 Struktur Primer subunit
Interaksi dengan FAD Ikatan disulfida Interaksi dengan Substrat

8 Inhibisi Ag+, Hg2+, Cu2+ dapat menjadi inhibitor melalui pembentukan kompleks secara non kompetitif (Toren and Burger 1968) Inhibisi oleh substrat (glukosa) dapat terjadi ketika kadar oksigen rendah (Nicol dan Duke, 1966) Akumulasi produk (H2O2 dan D- gluconolactone) juga dapat mengihibisi reaksi (Kleppet, 1966, Bao et al.2001) Silver(I) has been determined in the range of to 0.2 μg/ml with an absolute error of less than 14 ng. Mercury(II) has been determined in the range of 0.1 to 0.4μg/ml with a maximum error of 34 ng. Below a concentration of 260 μg/ml Pb(II), no appreciable inhibition was observed. In the glucose reaction, reduction of the flavin is very fast, the second reduced form being formed slowly; thus, at high oxygen levels, the reaction producing the second reduced form cannot compete with oxygen for the first reduced form. At low oxygen levels, the reaction of the first reduced form with glucose to produce the second becomes important. Both hydrogen peroxide (Eq. 2) and D-gluconolactone (Eq. 3) breaks down spontaneously and catalytically. Despite this, GOX’s enzymatic activity is reduced when hydrogen peroxide accumulates and inactivates the enzyme (Kleppet 1966); the breakdown product of D-gluconolactone, gluconic acid (C6H12O7) accumulates, reducing pH of the solution. Not surprisingly, both gluconic acid (Miron et al. 2004) and hydrogen peroxide (Bao et al. 2001, 2003) can result in product inhibition of GOX.

9 Aplikasi Penentuan kadar glukosa dengan penggunaan biosensor (Wilson and Turner, 1992). GOx, bersama dengan catalase, digunakan pada berbagai industri makanan dan minuman, seperti dalam pembuatan bir, soft drinks, roti, mayonaise, makanan kaleng dll untuk menstabilkan warna dan rasa dengan penghilangan oksigen (Crueger and Crueger, 1990). Gox juga digunakan untuk produksi asam glukonat yang banyak digunakan dalam industri logam, beton, makanan, kosmetik, obat- obatan dll (Ramachandran et al. 2006). Commercial diagnostic kits for the determination of glucose in blood, serum and plasma are supplied commercially in colorimetric diagnostic kits (Wilson and Turner, 1992). The use of GOX has found application in the textile industry as a method for producing hydrogen peroxide for bleaching (Tzanov et al., 2002). Tzanov et al. (2002) covalently immobilised GOX on alumina and glass supports, resulting in higher recoveries. Maximum hydrogen peroxide concentrations of 0.35 gL-1 and 0.24 gL-1 were reached after 450 minutes for GOX immobilised on the glass and alumina supports respectively (20g glucose in 50ml 0.1 M acetate buffer, pH 5, 35°C, and aerated at 5 L/min). The alumina support proved more stable at the 17 operational conditions and could be used for 3 consecutive runs. The hydrogen peroxide produced was tested for bleaching scoured woven cotton fabric and was found to be comparable to standard bleaching processes. No stabilisers were needed since the gluconic acid produced acted as a stabilising agent.

10 Aplikasi di Industri makanan
Menghilangkan oksigen dalam pembuatan mayonaise dan pengalengan makanan Meningkatkan kualitas textur roti Untuk mengendalikan pencoklatan (browning) pada pemrosesan dan penyimpanan pasta dari buah Bersama lactoperoxidase digunakan sebagai pengawet/agen antimikroba pada industri berbahan dasar susu GOX together with CAT or HRP has a range of applications in the food industry for glucose determination and as an antioxidant. Below is a reaction scheme for the GOX-CAT enzyme system: The GOX-CAT enzyme system was used by Isaksen and Adler-Nissen (1997) to scavenge oxygen in mayonnaises with different oxidative susceptibility. The investigation proved that the GOX-CAT enzyme system could be used to retard the lipid oxidation in mayonnaise stored at 5°C and 25°C, in mayonnaises containing pure soybean oil and where up to half the vegetable oil had been supplemented with fish oil. The enzyme system was responsible for scavenging the oxygen during glucose oxidation thereby decreasing the availability of the oxygen for lipid metabolism. Oxygen is known to be a key factor in the browning of fruit purees, and the enzyme system was shown to have the capability to control the non-enzymatic browning during fruit processing and purée storage. The scavenging of the oxygen by the enzyme system had a stabilising effect. The lactoperoxidase (LP) system, when used in conjunction with glucose oxidase, is a very useful anti-microbial agent. Lactoperoxidase system is part of the immune system’s innate defence mechanism against foreign micro-organisms and can be found in mammalian secretions such as milk, tears and saliva. This system consists of three components— lactoperoxidase, thiocyanate (SCN−) and hydrogen peroxide. LP system activation occurs only in the presence of thiocyanate and hydrogen peroxide. Catalysis by lactoperoxidase generates active intermediates, which has anti-microbial properties and is completely safe to humans. The presence of GOX and its substrate (glucose) allows hydrogen peroxide required by LP system to be continuously generated and replenished (Seifu et al. 2005).

11 Aplikasi di Industri Minuman
Produksi wine kadar alkohol rendah (Pickering et al. 1998) Reduction of the fermentation alcohol potential was achieved by pre-treating the grape juice with the GOX-CAT enzyme system to convert the available glucose to gluconic acid (Scheme 1.5). The low pH of the grape juice was determined to be a limiting factor, which was subsequently overcome by the use of calcium carbonate prior to the enzymatic treatment. A glucose conversion of 87% was achieved with this system. Glucose oxidase consumes some of the glucose present, making them unavailable for alcohol fermentation, thereby resulting in wine with reduced alcohol. At the same time, hydrogen peroxide generated may reduce the activity or growth of the S. cerevisiae used for alcohol fermentation.

12 Aplikasi di Bidang Kesehatan: Biosensor
Sensor glukosa darah dan cairan tubuh lainnya The potential applications of biosensors in daily life are tremendous: from environmental and water analysis to personalized healthcare, from quality food control to home patient monitoring," said Garrido. "In addition, other aspects with strong impact on public healthcare - such as drug screening and genetic disease control - will certainly benefit from biosensors

13 Latar belakang penelitian
GOx yang disekresikan pada S. cerevisiae menunjukkan hyperglycosylation yang tinggi dibandingkan dengan GOx dari A. niger Konstruksi ekspresi GOx menggunakan mutan S. cerevisiae yang mana gen PMR1 yang mengkode Ca2+ -ATPase ditiadakan sehingga dapat menekan hiperglikosilasi GOx Yeast mutations have already been made to alter their secretion properties in an attempt to enhance the secretion efficiency of heterologous proteins. As such, the increased secretion of bovine prochymosin has been achieved with the ssc1 mutant of S. cerevisiae [13]. The SSC1 gene was subsequently shown to be identical to the PMR1 gene encoding a P-type Caþþ-ATPase thereby implying that the increased secretion resulted from a defect in the Caþþ pump in the yeast secretory pathway [14]. The Caþþ-transporting ATPase in the Golgi of S. cerevisiae is known to strongly affect the glycosylation of proteins [18]. Yeast strains carrying pmr1D allele have also been shown to reduce the extent of hyperglycosylation in the secreted proteins [19].

14 Tujuan Melakukan ekspresi secretori dan pemurnian glukosa oksidase dari Apergillus niger pada S. cerevisiae Menguji pengaruh hiperglikosilasi dari GOx rekombinan terhadap properti enzim dan efisiensi sekresi To examine whether the hyperglycosylation of the recombinant GOD affected its enzyme properties and secretion efficiency, a S. cerevisiae mutant strain was manufactured in which the PMR1 gene encoding Caþþ-ATPase was disrupted and then the pmr1D mutant strain was transformed with the GOD expression plasmid pYGOD-His. Six consecutive histidine residues were fused to the C-terminus of the GOD to facilitate purification of the expressed protein using IMAC.

15 Metode Konstruksi mutan S. cerevisiae dan vektor ekspresi
Transformasi dengan plasmid pYGOD-His Kultivasi Pemurnian rekombinan Gox-His6 Immunobloting SDS-PAGE

16 Hasil: Immunoblot Intracellular Extracellular
When compared to the GODHis6 expressed in the wild-type strain, the GOD-His6 expressed in the pmr1D strain did not exhibit a broad diffuse band on SDS–PAGE. This indicates that the GOD-His6 expressed in the pmr1D strain was not hyperglycosylated. As observed in a previous report [3], the natural A. niger GOD (Sigma) was also not hyperglycosylated. M: marker; 1: Native GOx A. Niger; 1 ,2: Wild Type ; 3,4 Mutan type

17 Efek mutasi pada pertumbuhan sel dan sekresi Gox-His6
The secreted amount of GOD-His6 in the wild-type was slightly higher than that in the mutant strain. However, when taking the secreted amount of GOD-His6 per unit cell mass into account, the pmr1D mutant strain secreted a higher amount of GOD-His6 than the wild-type. Yet, the final level of GOD-His6 secreted into the extracellular medium was not improved due to the decreased cell mass of the pmr1D mutant strain.

18 Pemurnian During a gradual increase in the imidazole concentration, a sharp protein peak was observed in the chromatogram (Figs. 2 and 3). This fraction was selectively collected and analyzed by SDS–PAGE.

19 Pemurnian dan SDS-Page
M: Marker 1: GOx pada Wild Type 2: GOx pada Mutan Type the GOD-His6 was purified to homogeneity on SDS–PAGE by single-step IMAC. As expected, recombinant GOD-His6 purified from the culture medium of the pmr1D mutant strain was not hyperglycosylated, whereas GOD-His6 from the culture medium of the wild-type migrated as a broad diffusion band due to extensive N-linked hyperglycosylation. The purification results are summarized in Table 2. The specific activities of the GOD-His6 purified from the culture media of the wild-type and pmr1D mutant strains were 189 U/mg of protein and 194 U/mg of protein, respectively. According to a previous report [3], the specific activities of natural A. niger and yeast-derived recombinant GODs on a protein basis are very similar. When combining these results, this reconfirms that the additional carbohydrate moiety of GOD has a minimal affect on the catalytic efficiency of the enzyme.

20 Pengaruh pH GOx A. Niger GOx-His6 mutan type Gox-His6 hiperglikosilasi
The optimal pH was found to be 6.0 for all these three enzymes. As observed in a previous report [3], natural A. niger GOD exhibited a narrower pH-stability profile than the hyperglycosylated GOD-His6. In contrast, the pH-stability profile of the natural GOD was very similar to that of the pmr1D mutant-derived GOD-His6.

21 Pengaruh Temperatur GOx A. Niger GOx- His6 mutan type
GOx – His6 hiperglikosilasi The thermal stabilities of the natural GOD and pmr1D mutant-derived GOD-His6 were very similar and less thermally stable than the hyperglycosylated GOD-His6.

22 Kesimpulan Properti fisik dan efisiensi katalitik dari GOx-His6 mutan pmr1Δ hampir sama dengan GOx native dari A. niger Delesi gen PMR1 pada S. cerevisiae dapat meningkatkan efisiensi sekresi GOx namun menganggu pertumbuhan sel

23 Terima kasih