Coriolopsis Polyzona Mucl 38443'ten Lakkaz Kodlayan Bir Cdna'nın Klonlanması Ve Pichia Pastoris’te İfade Edilmesi
Coriolopsis Polyzona Mucl 38443'ten Lakkaz Kodlayan Bir Cdna'nın Klonlanması Ve Pichia Pastoris’te İfade Edilmesi
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Tarih
2017-02-22
Yazarlar
Pinar, Orkun
Süreli Yayın başlığı
Süreli Yayın ISSN
Cilt Başlığı
Yayınevi
Fen Bilimleri Enstitüsü
Institute of Science and Technology
Institute of Science and Technology
Özet
Lakkazlar (EC 1.10.3.2), birçok farklı mantar, bitki, bakteri ve böcek türlerinden elde edilebilen, fenolik olan ve fenolik olmayan lignin içerikli bileşiklerin ve çevre kirleticilerin oksidasyonunu katalize edebilen, çoklu bakır oksidazların protein süper ailesinin bir üyesidir. Bugüne kadar 120’den fazla lakkaz enzimi fungal kültürlerden izole edilerek, biyokimyasal ve katalitik özellikleri aydınlatılmıştır. Bu noktada, tıbbi tanı kitleri, biyoremediasyon (pestisit ve herbisit gibi maddelerin temizlenmesi), endüstriyel atıkların detoksifiye edilmesi (kâğıt, tekstil ve petrokimya endüstrilerinden kaynaklı), içeceklerin işlenmesi (şarap, meyve suyu ve bira), şeker pancarı pektininden jel oluşturulması, askorbik asit belirlenmesi, biyosensör yapısında belirleme için kullanılması ile kozmetik ve ilaç malzemesi olarak kullanılması gibi farklı biyoteknolojik prosesler açısında önem taşımaktadırlar. Lignoselülozların parçalanması dünyanın ekosisteminde, özellikle orman ekosistemlerinde yer alan organik karbonların yüzde beşini oluşturan ölü odunsu yapılardan sağlanan karbon geri kazanımının gerçekleşmesi için kritik bir öneme sahiptir. Küresel karbon döngüsü için çok önemli bir rol oynayan lignoselülozik kompleksleri degrede edebilme özelliğine sahip çoklu enzim sistemine sahip tek mikroorganizma tipi beyaz çürükçül mantarlardır. Özellikle beyaz çürükçül Basidiyomisetler, tüm odunsu lignoselüloz bileşiklerinin yumuşak ve sert odunsu kısımlarının çürütülmesini sağlayan hücre dışına salgılanan lakkaz benzeri özel oksidatif enzimler üretirler. Askomiset ve Döteromisetler ile karşılaştırıldığında, beyaz çürükçül Basidiyomisetler ligninin parçalanması konusunda ön plana çıkmaktadır. Collybia velutipes, Fomes annosus, Fomes fomentarius, Lentinus edodes, Phanerochaete chrysosporium, Pholiota mutabilis, Pleurotus ostreatus, Poria subacida, Sporotrichum pulverulentum, Trametes sanguinea ve Trametes versicolor türleri bilinen en iyi lakkaz üreticileri ve lignin parçalayıcılarını oluşturmaktadır. Basidiomycete Coriolopsis polyzona, Coriolaceae ailesine ait beyaz bir çürük mantar olup, yüksek verimli bir ligninolitik enzim üreticisidir. Heterolok ekspresyon sistemlerinin kurulması, enzim özelliklerinin iyileştirilmesini sağlayacak, protein mühendisliği çalışmalarının önünü açacak önemli bir basamaktır. Ayrıca, farklı kaynaklardan yeni lakkazların elde edilmesini kapsayan çalışmalar, endüstride yeni kullanım alanine sahip ve/veya düşük maliyetli yeni lakkazların yakalanmasını sağlaması açısından, önemli bulunmaktadır. Bu araştırmada, Coriolopsis polyzona MUCL 38443 suşundan izole edilen ve Cplcc1 olarak adlandırılan yeni bir lakkaz genine ait cDNA, Pichia pastoris’de heterolok olarak eksprese edilmiş ve karakterizasyonu gerçekleştirilmiştir. Öncelikle, Coriolopsis polyzona MUCL 38443 ait, kısmi lakkaz cDNA kütüphanesi kurulmuştur. Bu kütüphaneden yaklaşık 52 klona ait parça lakkaz nükleotit dizileri elde edilmiş, birbirleri ile benzerlikleri ClustalW2 ile “Principal Coordinates Analysis (PCoA) pogramları ile karşılaştırılmıştır. Sonuç olarak kurulan kısmi lakkaz cDNA kütüphanesinde, sadece 3 farklı kısmi lakkaz cDNA dizisinin bulunduğu belirlenmiş ve Cplcc1, Cplcc2 ve Cplcc3 olarak isimlendirilmişlerdir. Bulunan bu kısmi lakkaz cDNA dizileri, Ulusal Biyoteknoloji Bilgi Merkezi (NCBI) veri tabanına önceden yüklenmiş olan ve lakkaz kodlayan diziler ile karşılaştırıldığında, yaklaşık % 80 benzerlik gösterdiği belirlenmiştir. Oluşturulmuş olan kısmi cDNA kütüphanesinde, belirlenen bu kısmi üç cDNA dizisi arasında da, en sık Cplcc1 cDNA dizisine rastlanmıştır. Bu sonuca dayanarak, Cplcc1 lakkaz geninin, Coriolopsis polyzona MUCL 38443’de en baskın ifade edilen lakkaz geni olduğu anlaşılmıştır. Cplcc1 geninin 1563 bç uzunluğuna sahip ve 520 amino asit kodlayan bir açık okuma çerçevesi ile 20 amino asitlik sinyal peptitini içerdiği belirlenmiştir. Cplcc1 cDNA dizisi “Trametes hirsuta laccase A mRNA” dizisi ile % 82 benzerlik gösterirken, Cplcc1 genine ait çıkarılmış amino asit dizisi ise benzer şekilde “Trametes hirsuta laccase A” (AIZ72721.1) ile % 88 benzerlik göstermiştir. CpLcc1 lakkazına ait moleküler ağırlık değeri, teorik moleküler ağırlık hesaplama aracı ile yaklaşık 54 kDa olarak (Compute pI/MW, ExPasy) hesaplanmıştır. Buna ek olarak, Cplcc1 geninin boyutu 2106 bç uzunluğunda ve % 57 GC içeriğine sahip olduğu, 51-58 bç uzunluğunda on adet intron ve beş adet potansiyel N-glikosilasyon bölgesini içerdiği tespit edilmiştir. Bununla birlikte, Cplcc1 geni Trametes hirsuta lakkaz geni (Lac) (EU492907.1) ile % 76 benzerlik göstermiştir. Cplcc1 cDNA ve genomik DNAsına ait nükleotit sekansları GenBank veri tabanına sırasıyla KT802746 ve KT802747 erişim numarası ile kaydedilmiştir. Ayrıca, tam boyuttaki Cplcc1 cDNAsı maya mekik ifade vektörü olan pPICZC’ye kendi sinyal dizisi ile birlikte başarılı bir şekilde klonlanmış ve metilotrofik bir maya olan Pichia pastoris’te eksprese edilmiştir. En iyi kültür koşullarını belirlemek amacıyla, metanol konsantrasyonu, bakır konsantrasyonu, L-Alanin konsantrasyonu ve büyüme sıcaklığının etkisi araştırılmıştır. Optimizasyon çalışmaları sonucunda, optimum koşullar, BMGY/BMMY besiyeri içerisinde % 1,5 metanol, 0.8mM CuSO4 ve % 0,6 L-Alanin eklenerek, 250 rpm ve 28°C olarak belirlenmiştir. Elde edilen en yüksek rekombinant CpLcc1 lakkaz aktivitesi 800 U l-1 olarak bulunmuş ve optimizasyon çalışmaları boyunca lakkaz aktivitesi 9,8 kat arttırılabilmiştir. Optimizasyon çalışmaları sonrasında, rekombinant lakkazın kısmi olarak saflaştırılması, amonyum sülfat çöktürmesi, Q-sepharose anyon değişim ve Superdex-75 boyut kromatografisi adımları ile gerçekleştirilmiştir. Kısmi saflaştırılmış rekombinant CpLcc1 lakkazına ait rölatif moleküler ağırlık değeri SDS-PAGE analizi ile, teorik moleküler ağırlık değeri ile benzer biçimde 54kDa olarak ayrıca bulunmuştur. Bu değer önceden raporlanmış çalışmalarla uygunluk göstermektedir. Rekombinant lakkaz aktivitesine ait maksimum değer 2,2’-azino-bis (3-etilbenzotiazolin-6-sulfonik asit) (ABTS) substratı için optimum sıcaklık olan 70°C ve optimum pH 3.0’de gözlemlenmiştir. Diğer yandan rekombinant CpLcc1 30°C’de bir haftadan fazla stabil kalabilirken, yarılanma ömrü ise 8 saatten yüksektir. Ayrıca 40°C ve 50°C’deki yarılanma süresi sırasıyla 8 saat ve 30 dakika iken, enzim aktivitesi 60°C, 70°C ve 80°C’lerde ise dakikalar içerisinde ani düşüş göstermektedir. Km, Vmax, kcat ve kcat Km-1 değerleri sırasıyla 0,137 mM, 288,6 μmol d-1 l-1, 5.73×105 d-1 and 4.18×106 d-1 mM-1, olarak hesaplanmıştır. Sodyum azid, L-sistein ve SDS maddeleri ise olağan inhibitörler olarak belirlenmiştir. Sonuç olarak, bu çalışma ile ilk kez, Coriolopsis polyzona MUCL 38443 suşunda ifade edilen lakkaz genleri belirlenmiş, bunların arasından CpLcc1 olarak adlandırılan lakkaz enziminin P. pastoris mayasında heterolok üretimi, kısmi saflaştırılması başarı ile gerçekleştirilmiş ve biyokimyasal karakterizasyonu tamamlanmıştır.
Laccases (EC 1.10.3.2) are members of the protein superfamily of multi-copper oxidases isolated from various fungi, plants, bacteria and insects that are able to catalyze the oxidation of non-phenolic and phenolic lignin related compounds and environmental pollutants. Up to date, over 120 laccases have been isolated from fungal cultures. Their biochemical and catalytic properties have been identified. They are important for several biotechnological processes such as the medical diagnosis, bioremediation (to clean up pesticides, herbicides etc.), the detoxification of industrial effluents (from paper, pulp, textile and petrochemical industries), the processing of the beverages (wine, fruit juice and beer), the gelation of the sugar beet pectin, the determination of the ascorbic acid, baking, detection processes as biosensors and being as ingredients in cosmetics and pharmaceutical area. Lignocellulose degradation is a critical process for carbon recycling of earth’s ecosystems and particularly dead wood that represents globally 5 % of organic carbon in the forest ecosystems. White-rot fungi are the only microorganisms able to degrade lignocellulosic complex by a multi-enzyme system which plays very important role in the global carbon cycle. Especially, the white rot Basidiomycetes are able to decay all wood lignocellulose components, both in softwood and hardwood, as they produce unique extracellular oxidative enzymes such as laccases. The white-rot Basidiomycetes have been a focus for lignin degradation when compared to Ascomycetes and Deuteromycetes. Collybia velutipes, Fomes annosus, Fomes fomentarius, Lentinus edodes, Phanerochaete chrysosporium, Pholiota mutabilis, Pleurotus ostreatus, Poria subacida, Sporotrichum pulverulentum, Trametes sanguinea and Trametes versicolor are Basidiomycetes known as best laccase producer and lignin degraders. Basidiomycete Coriolopsis polyzona is a white rot fungus belonging to the family of Coriolaceae and a promising high-yield ligninolytic enzyme producer. In the present research, a novel laccase gene, named as Cplcc1, and its corresponding cDNA were isolated from the Coriolopsis polyzona MUCL 38443 strain, heterologously expressed and characterized. Fundamentally, a partial laccase cDNA library of C. polyzona MUCL 38443 was constructed. Three distinct partial laccase cDNA sequences were identified and described as Cplcc1, Cplcc2 and Cplcc3. These laccase specific cDNA sequences showed 80 % similarity with previously submitted laccase encoding sequences in National Center for Biotechnology Information (NCBI) database. In the partial cDNA library, the dominant cDNA sequences were belonging to Cplcc1, which appeared to be the major expressed laccase gene in Coriolopsis polyzona MUCL 38443. The Cplcc1 gene consists of a 1563 bp open reading frame encoding a 520 amino acids protein with a 20-residue putative signal peptide. The molecular weight of the CpLcc1 laccase protein was determined as 54 kDa with theoretical MW (Compute pI/MW, ExPasy) calculator using full-length deduced amino acid sequence. While the complete cDNA sequence of Cplcc1 indicates 82 % similarity with Trametes hirsuta laccase A mRNA, the deduced amino acid sequence of Cplcc1 also revealed 88 % identity with Trametes hirsuta laccase A (AIZ72721.1). In addition, the Cplcc1 gene was also sequenced and characterized. The size of the Cplcc1 gene is 2106 bp with 57 % GC content and interrupted with ten introns ranging from 51-58 bp and contains five potential N-glycosylation sites. Thus, Cplcc1 gene has 76 % identity with Trametes hirsuta laccase (Lac) gene (EU492907.1). The nucleotide sequences of cDNA and genomic DNA sequences of Cplcc1 have been deposited in the GenBank database under accession number KT802746 and KT802747, respectively. Furthermore, the full-length Cplcc1 cDNA was successfully cloned into the yeast shuttle expression vector pPICZC with their own secretion signal sequence and expressed in methylotrophic yeast Pichia pastoris. To optimize the recombinant laccase production, optimum cultivation conditions including the effect of methanol concentration, copper concentration, L-Alanine concentration and growth temperature were investigated. As a result of optimization study, BMGY/BMMY medium supplemented with 1.5 % methanol, 0.8 mM CuSO4 and 0.6 % L-Alanine with 250 rpm and 28°C were obtained as optimum conditions. The maximum recombinant CpLcc1 laccase activity was found as 800 U l-1. As a result of optimization studies, laccase activity was increased approximately 9.8 fold. Then, recombinant laccase was partially purified by ammonium sulfate precipitation, Q-sepharose anion exchange and Superdex-75 gel filtration chromatography. Based on the SDS-PAGE, the relative molecular mass of the partially purified recombinant laccase CpLcc1 was estimated as 54 kDa with the SDS-PAGE analysis which is consistent with theoretical MW and in the range reported for most of the fungal laccases. The maximum recombinant laccase activity was observed for 2,2’-azinobis-(3-ethylbenzthiaoline-6-sulfonic acid) (ABTS) as the substrate with optimum temperature as 70°C and pH value as 3.0. On the other hand, recombinant CpLcc1 was stable at 30°C more than a week and t1/2 were higher than 8 hours. While half-life of CpLcc1 at 40°C for 8 hours and at 50°C for 30 minutes, activity decreased dramatically in few minutes at 60°C, 70°C and 80°C. Km, Vmax, kcat and kcat Km-1 values of the recombinant laccase were identified as 0.137 mM, 288.6 μmol min-1 l-1, 5.73×105 min-1 and 4.18×106 min-1 mM-1, respectively. Sodium azide, L-cysteine and SDS were found as usual inhibitors. Present study is the first report on the cloning and the characterization of a laccase gene and its encoding laccase from the white rot fungus strain Coriolopsis polyzona MUCL 38443.
Laccases (EC 1.10.3.2) are members of the protein superfamily of multi-copper oxidases isolated from various fungi, plants, bacteria and insects that are able to catalyze the oxidation of non-phenolic and phenolic lignin related compounds and environmental pollutants. Up to date, over 120 laccases have been isolated from fungal cultures. Their biochemical and catalytic properties have been identified. They are important for several biotechnological processes such as the medical diagnosis, bioremediation (to clean up pesticides, herbicides etc.), the detoxification of industrial effluents (from paper, pulp, textile and petrochemical industries), the processing of the beverages (wine, fruit juice and beer), the gelation of the sugar beet pectin, the determination of the ascorbic acid, baking, detection processes as biosensors and being as ingredients in cosmetics and pharmaceutical area. Lignocellulose degradation is a critical process for carbon recycling of earth’s ecosystems and particularly dead wood that represents globally 5 % of organic carbon in the forest ecosystems. White-rot fungi are the only microorganisms able to degrade lignocellulosic complex by a multi-enzyme system which plays very important role in the global carbon cycle. Especially, the white rot Basidiomycetes are able to decay all wood lignocellulose components, both in softwood and hardwood, as they produce unique extracellular oxidative enzymes such as laccases. The white-rot Basidiomycetes have been a focus for lignin degradation when compared to Ascomycetes and Deuteromycetes. Collybia velutipes, Fomes annosus, Fomes fomentarius, Lentinus edodes, Phanerochaete chrysosporium, Pholiota mutabilis, Pleurotus ostreatus, Poria subacida, Sporotrichum pulverulentum, Trametes sanguinea and Trametes versicolor are Basidiomycetes known as best laccase producer and lignin degraders. Basidiomycete Coriolopsis polyzona is a white rot fungus belonging to the family of Coriolaceae and a promising high-yield ligninolytic enzyme producer. In the present research, a novel laccase gene, named as Cplcc1, and its corresponding cDNA were isolated from the Coriolopsis polyzona MUCL 38443 strain, heterologously expressed and characterized. Fundamentally, a partial laccase cDNA library of C. polyzona MUCL 38443 was constructed. Three distinct partial laccase cDNA sequences were identified and described as Cplcc1, Cplcc2 and Cplcc3. These laccase specific cDNA sequences showed 80 % similarity with previously submitted laccase encoding sequences in National Center for Biotechnology Information (NCBI) database. In the partial cDNA library, the dominant cDNA sequences were belonging to Cplcc1, which appeared to be the major expressed laccase gene in Coriolopsis polyzona MUCL 38443. The Cplcc1 gene consists of a 1563 bp open reading frame encoding a 520 amino acids protein with a 20-residue putative signal peptide. The molecular weight of the CpLcc1 laccase protein was determined as 54 kDa with theoretical MW (Compute pI/MW, ExPasy) calculator using full-length deduced amino acid sequence. While the complete cDNA sequence of Cplcc1 indicates 82 % similarity with Trametes hirsuta laccase A mRNA, the deduced amino acid sequence of Cplcc1 also revealed 88 % identity with Trametes hirsuta laccase A (AIZ72721.1). In addition, the Cplcc1 gene was also sequenced and characterized. The size of the Cplcc1 gene is 2106 bp with 57 % GC content and interrupted with ten introns ranging from 51-58 bp and contains five potential N-glycosylation sites. Thus, Cplcc1 gene has 76 % identity with Trametes hirsuta laccase (Lac) gene (EU492907.1). The nucleotide sequences of cDNA and genomic DNA sequences of Cplcc1 have been deposited in the GenBank database under accession number KT802746 and KT802747, respectively. Furthermore, the full-length Cplcc1 cDNA was successfully cloned into the yeast shuttle expression vector pPICZC with their own secretion signal sequence and expressed in methylotrophic yeast Pichia pastoris. To optimize the recombinant laccase production, optimum cultivation conditions including the effect of methanol concentration, copper concentration, L-Alanine concentration and growth temperature were investigated. As a result of optimization study, BMGY/BMMY medium supplemented with 1.5 % methanol, 0.8 mM CuSO4 and 0.6 % L-Alanine with 250 rpm and 28°C were obtained as optimum conditions. The maximum recombinant CpLcc1 laccase activity was found as 800 U l-1. As a result of optimization studies, laccase activity was increased approximately 9.8 fold. Then, recombinant laccase was partially purified by ammonium sulfate precipitation, Q-sepharose anion exchange and Superdex-75 gel filtration chromatography. Based on the SDS-PAGE, the relative molecular mass of the partially purified recombinant laccase CpLcc1 was estimated as 54 kDa with the SDS-PAGE analysis which is consistent with theoretical MW and in the range reported for most of the fungal laccases. The maximum recombinant laccase activity was observed for 2,2’-azinobis-(3-ethylbenzthiaoline-6-sulfonic acid) (ABTS) as the substrate with optimum temperature as 70°C and pH value as 3.0. On the other hand, recombinant CpLcc1 was stable at 30°C more than a week and t1/2 were higher than 8 hours. While half-life of CpLcc1 at 40°C for 8 hours and at 50°C for 30 minutes, activity decreased dramatically in few minutes at 60°C, 70°C and 80°C. Km, Vmax, kcat and kcat Km-1 values of the recombinant laccase were identified as 0.137 mM, 288.6 μmol min-1 l-1, 5.73×105 min-1 and 4.18×106 min-1 mM-1, respectively. Sodium azide, L-cysteine and SDS were found as usual inhibitors. Present study is the first report on the cloning and the characterization of a laccase gene and its encoding laccase from the white rot fungus strain Coriolopsis polyzona MUCL 38443.
Açıklama
Tez (Doktora) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2017
Thesis (Ph.D.) -- İstanbul Technical University, Institute of Science and Technology, 2017
Thesis (Ph.D.) -- İstanbul Technical University, Institute of Science and Technology, 2017
Anahtar kelimeler
Coriolopsis Polyzona,
Enzim Aktivitesi,
Heterolok Ekspresyon,
Lakkaz,
Pichia Pastoris,
Saflaştırma,
Coriolopsis Polyzona,
Enzyme Activity,
Heterologous Expression,
Laccase,
Pichia Pastoris,
Purification