Development Of Novel Biocatalysts For Industrial Applications

Tez (Doktora) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2012Thesis (PhD) -- İstanbul Technical University, Institute of Science and Technology, 2012Küresel ısınma odaklı tartışmalar ve bu konuda oluşan kaygılar son yıllarda bilim insanları ve toplum içinde giderek artmaktadır. Bu durum, petrol kaynaklı üretimler yerine yenilenebilir biyolojik kaynaklara yönelimi teşvik etmektedir. “Yeşil proses” olarak adlandırılabilecek biyokatalizörlerin farklı endüstriyel alanlarda kullanımının önemi yakın zamanda idrak edilmiştir. Kimyasal katalizörler yerine hayvansal, bitkisel ve bakteriyel kaynaklardan elde edilen enzimlerin kullanımı endüstriyel proseslerde çeşitli avantajlar sağlamaktadır. Kiral merkezli moleküllerin sentezi kimyasal yöntemlerle gerçekleştirildiğinde rasemik karışımlar elde edilmektedir. Enzimatik reaksiyonlar ise enantiomerik saflıkta ürün üretimine imkan verdiklerinden, biyokatalizörlerin kiral bileşik üretiminde kullanılması çok uygundur. Biyokatalizör kullanımının diğer avantajları enzimlerin biyobozunur olmaları, katalizledikleri reaksiyonda stereo-seçici, bölgesel-seçici ve kimyasal-seçici davranmaları, görece düşük sıcaklıkta, ılıman pH ve basınç koşullarında fonksiyon göstermeleri ve çevre dostu olmaları şeklinde sıralanabilir. Bu avantajlarından dolayı biyokatalizörlerin tıp, kimya, gıda üretimi ve tarım endüstrilerinde kullanım alanları giderek artmaktadır. Fakat doğal çevreden izole edilen enzimlerin doğrudan endüstriyel proseslerde kullanılabilmeleri herzaman mümkün olmamaktadır. Enzim karakteristik özelliklerinin (aktivite, stabilite, çözünürlük, substrat ve koenzim seçiciliği ve optimum pH aralığı) sıklıkla ilgili endüstriyel üretime göre optimize edilmesi gerekmektedir. Protein mühendisliği yöntemleri, ilgili proses koşullarına uygun enzimlerin dizayn edilmesine olanak tanımaktadır. Protein mühendisliği, moleküler biyoloji tekniklerinin kullanılması yolu ile enzimlerin yapı ve fonksiyonlarının moleküler düzeyde anlaşılıp, genetik yapılarının değiştirilerek, istenilen yapı ve fonksiyona sahip yeni enzimlerin üretilmesini mümkün kılmaktadır. Burada, endüstriyel öneme sahip olan Bacillus stearothermophillus laktat dehidrogenaz (bsLDH) ve Rhodotorula graminis fenilalanin liyaz (rgPAL) olarak adlandırılan enzimlerin substrat özgüllüklerinin iyileştirilmesini hedefledik. Bu iki enzimin özelliklerinin iyileştirilmesi için protein mühendisliğinin üç yöntemi uygulanmıştır; DNA shuffling (DNA karıştırma), bölgeye özel mutasyon yaklaşımları ile kombinatoryal saturasyon aktif bölge testi (CASTing). bsLDH’ın substrat spesifikliği, DNA karıştırma ve bölgeye özel mutasyon yaklaşımları ile başarıyla değiştirilmiştir. rgPAL’ın aktivitesi ise seçilen farklı substratlara karsı CASTing yöntemi kullanılarak arttırılmıştır. Bu tez kapsamında yeni bsLDH ve rgPAL proteinlerinin üretilmesine yönelik farklı stratejiler detaylı bir şekilde incelenmiştir. Üç deneysel bölümden oluşan tezin ilk iki bölümü bsLDH’ın düzenlenmesini tanımlarken, son bölüm rgPAL için CASTing metodunun uygulanmasını açıklamaktadır. bsLDH, okzo-asitlerin (pirüvat) hidroksi asitlere (laktat) karşılıklı dönüşüm reaksiyonunu NADH/NAD+ çiftini redoks kofaktörü olarak kullanarak katalizler. Enzim, eczacılık ve tarım alanında anahtar role sahip kiral moleküllerin sentezinde kullanılan ara ürünlerin üretiminde kullanıldığından ticari öneme sahiptir. İlgili okzo-asitlerin kiral hidroksi asitlerinin yüksek stereokimyasal seçicilikle üretilmesine imkan vermesine rağmen; bsLDH enziminin en önemli dezavantajı son derece sınırlı substrat özgüllüğüne sahip olmasıdır. Tezin ilk kısmında; bsLDH’ın substrat seçiciliğini değiştirmek için DNA karışım yöntemi kullanılmıştır. DNA karışım yöntemi ile elde edilen rekombinant mutant bsLDH kolonilerinden, malatın oksidasyonunu katalizleyebilenler uygun tarama ve seçme test yöntemi kullanılarakdiğerlerinden ayırt edilmişlerdir. Yönlendirilmiş evrim sonucunda sekiz (8) tane amino asit değişimine - Asn4Gln, Ser19Thr, Gln86Arg, Thr91Ser, Ala173Val, Glu183Asp,Gln221Asn ve Val267Thr- sahip olan mutant bsLDH proteininin substrat seçiciliğini pirüvat/laktatdan okzaloasetat/malata değiştirmiştir. Bahsedilen sekiz mutasyonun bsLDH’de malat dehidrogenaz aktivitesi oluşumuna neden olduğu ve malatın oksidasyonunu, pirüvata kıyasla 1009 kat daha hızlı olacak şekilde gerçekleştirdiği görülmüştür. Bu sonuç, yapı bilgisi kullanılarak yüksek verimli tekli mutasyonlarla hedeflenenin gerçekleştirildiği tasarımlara göre, yönlendirilmiş evrimin de özgüllüklerde yüksek değişikliklere yol açabileceğini göstermiştir. Tezin ikinci kısmında; X-ışını kristal yapısı ve biriken literatür bilgisi kullanılarak bsLDH rasyonel olarakmandelik asitle reaksiyona girecek şekilde dizayn edilmiştir. Mandelat dehidrogenaz aktivitesi, InsightII moleküler modelleme programı kullanılarak; bsLDH enziminde oluşturulmuştur. Modelleme programı enerji minimizasyonu sonucunda; Thr246Gly ve Ile240Ala mutasyonları ile bsLDH enziminin L-mandelik asidi substrat olarak kullanabileceği gösterilmiştir. Mutanlar oluşturulmuş ve mutant gen E.coli‘ye aktarılarak sentezi gerçekleştirilmiştir. Ayrıca mutant ve doğal bsLDH‘i saflaştırmak için TAGZyme saflaştırma sistemi oluşturulmuştur. Rasyonel olarak tasarlanan bsLDH’ın aktivitesi mandelat varlığında ölçülmüştür. Enzim kinetiği sonuçları, bu iki mutasyonun, yüksek saflıkta elde edilen mutant bsLDH’ın substrat özgüllüğünü laktatdan L-mandelik asite dönüştürdüğünü göstermiştir. Tezin son kısmında; E.coli’ye ekspresyon vektörüyle klonlanmış Rhodotorula graminis kaynaklı fenilalanin amonyum liyaz (rgPAL) enziminin doğal olmayan amino asitlere karşı aktivite kazandırılmasını amaçladık. PAL, oksijensiz ortamda L-fenilalaninin trans-sinamik aside dönüşümünü katalizlemektedir. Bu reaksiyonun geri dönüşüm reaksiyonu, farmakoper olarak birçok peptidomimetrik ilaç molekülünün üretiminde L-fenilalanin analoglarının sentezinde kullanıldığından, enzim kimya endüstrisinde özel bir öneme sahiptir. Yarı rasyonel dizayn olarak değerlendirilen CASTing metodu çerçevesinde, her bir kütüphane için en az iki tane amino asit içeren beş kütüphane oluşturulmuştur. NRT yıkıcı kodon olarak bütün kütüphaneler için kullanılmıştır. 2000 koloni, geliştirilen protokol 1 ile taranmıştır. Birçok tarama adımından sonra seçilen substrata karşı yüksek aktivite gösteren dört rgPAL varyantı belirlenmiştir. PAL varyantlarının tanımlanmasını kolaylaştırmak için iki tane test yöntemi geliştirilmiştir. Birinci test yöntemi, elde edilen kütüphaneleri taramak ve oluşan trans sinamik asit (t-CA) miktarını ölçmek için uygulanmıştır. İkinci test yönteminde ise, belirleyici enzim olarak kullanılan oksidaz enzimi, PAL enzimleri tarafından üretilen fenilalaninin miktarının niceleyici olarak ölçülmesine olanak tanıyacak şekilde eşleştirilmiştir. Uygun saflaştırma prosesleri sonrasında, mutant bsLDH ve rgPAL enzimleri kinetik olarak karakterize edilmişlerdir. Enzim kinetiği çalışmaları, bsLDH ve rgPAL enzimlerinin substrat özgüllüklerinin başarılı bir şekilde değiştirildiğini göstermiştir. Elde edilen bu sonuçlar, uygun strateji uygulandığında protein mühendisliğinin, enzimlerin yeniden tasarlanmasında çok güçlü bir teknoloji olduğunu göstermiştir.Global warming and the resultant problems have made the scientific community and general public realize that there is a need to decrease dependency on oil for energy. It has also shown the need to increase the efficient use of energy in manufacturing and industrial processes. Green processes would be an alternative way of decreasing oil dependency and biotechnology is promoted as a way both to decrease oil dependency and as a source for renewable bio-based products. Biocatalysis (using enzymes or whole systems) can provide a valuable alternative to traditional chemical processes because it has many advantages such as; efficiency in enhancing the rate of chemical reactions and for their ability to discriminate between potential substrates. Therefore there is the possibility of using biological molecules to catalyse any reaction or modify any product of interest to industry. Biological catalysts from animal, plant and bacterial sources have evolved to perform most types of organic reactions, producing chirally pure and complex molecules with interesting biological properties. Biocatalysts have thus become important tools in medicine, the chemical industry, food processing and in agriculture. Industrial processes often require extreme conditions such as high pressure, temperature and extreme pH which require a large amount of energy to achieve and may produce unwanted toxic waste. Biological enzymes do not require such conditions and produce chirally pure products often without the disadvantages of unwanted toxic by products. The use of naturally occurring enzymes is not that common because of limited substrate and coenzyme specificity and kinetic problems with the reactions. Protein engineering is a promising approach which can be used to create enzymes with the desired properties. Enzymes can also be engineered to better understand the molecular basis for their functions so that they will be able to synthesize novel products in non-native environments. Here, we have aimed to improve the substrate specificities of two industrially important enzymes namely; Bacillus stearothermophillus lactate dehydrogenase (bsLDH) and Rhodotorula graminis phenylalanine amonyum lyase (rgPAL). To improve the properties of these two enzymes, we have applied three routes of protein engineering studies namely; DNA shuffling, site directed mutagenesis and combinatorial active site saturation (CASTing). The substrate specificity of bsLDH was successfully altered by using DNA shuffling and site directed mutagenesis approaches. The activity of rgPAL for the selected different substrate(s) was also increased by CASTing. This thesis details various strategies used to produce novel bsLDH and rgPAL proteins. It is divided into three experimental sections. The first two sections describe to engineer bsLDH and the last section explains the application of CASTing method on rgPAL. bslactate dehydrogenase catalyses the interconversion of an oxo-acid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD+ pair as a redox cofactor. The enzyme has a commercial significance as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. Although bsLDH allows the synthesis of chiral hydroxy acids from their corresponding oxo-acids with high stereochemical fidelity, this enzyme has the disadvantage of generally not having very broad substrate specificity. In the first part of the thesis; DNA shuffling was used to alter the substrate specificity of bsLDH. The recombinant colonies having shuffled bsLDH are blotted and screened for their ability to catalyse the oxidation of malate. The shuffled mutant LDH has eight amino acid substitutions -Asn4Gln, Ser19Thr, Gln86Arg, Thr91Ser, Ala173Val, Glu183Asp, Gln221Asn and Val267Thr- changing the substrate specificity of bsLDH from pyruvate / lactate to malate / oxaloacetate. The eight amino acid substitutions led to a new malate dehydrogenase that catalysed the oxidation of malate 1009 times faster than the pyruvate does. This result demonstrates that the blind shuffling can achieve a huge shift in specificity which was a known, highly effective single-site mutation designed using structural knowledge. In the second part of the thesis; we described attempts to change the reactivity of bsLDH towards L-mandelic acid. Using the Insight II molecular modelling program and protein engineering techniques, we have successfully introduced substantial mandelate dehydrogenase activity to the enzyme. Energy minimisation modelling studies suggested that two mutations, Thr246Gly and Ile240Ala, would allow the enzyme to utilise L-mandelic acid as a substrate. Genes encoding for the wild type and mutant enzymes were constructed, and the resulting bsLDH proteins were overexpressed in E. coli and purified using the TAGZyme system . Enzyme assays showed that insertion of this double mutation to the highly purified bsLDH switched the substrate specificity from lactate to L-mandelic acid. In the last part of the thesis, our aim was to increase the enzymatic activity of the phenylalanine ammonia-lyase (PAL) from Rhodotorula graminis for non-natural specific substrates. PAL catalyses the non-oxidative conversion of L-phenylalanine (L-Phe) to trans-cinnamic acid (t-CA). The reversibility of this reaction has been used to synthesise analogues of L-phenylalanine as pharmacophores in several peptidomimetic drug molecules and are therefore of particular interest to the fine chemical industry. Five (5) libraries were set up, with at least two residues in each library, around the active site based on CASTing method that is a semi rational design. The degenerate codon NRT was used for all libraries. A total of 2000 colonies were screened using a developed assay 1 and after several rounds of screening, four PAL variants which displayed activity against selected substrate. Two assays have been developed for facilitating the identification of PAL variants. The first one is applied for screening libraries and measuring the formation of t-CA during the deamination. In the second assay, an oxidase enzyme (as a reporterenzyme) is coupled for allowing the detection and quantification of phenylalanine production by PAL enzymes. Following the purification, the mutant bsLDH and rgPAL enzymes were kinetically characterised. Kinetic studies proved that the substrate specificities of bsLDH and rgPAL were successfully altered. These results imply that protein engineering is a powerful technology for enzyme redesign if an appropriate strategy is applied.DoktoraPh

Our experience of 200 patients: Usage and maintenance of long-term oxygen therapy and non-invasive ventilation devices at home [conferenceObject]

WOS: 000209370401808

Highly stable and reusable immobilized formate dehydrogenases: Promising biocatalysts for in situ regeneration of NADH

This study aimed to prepare robust immobilized formate dehydrogenase (FDH) preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150), Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU), and Immobead 150 support functionalized with aldehyde groups (FDHIALD). The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t1/2) of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 °C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH

Enhanced production of recombinant Staphylococcus simulans lysostaphin using medium engineering

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 degrees C. The production cost of 1000U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently

High-level heterologous expression of active <i>Chaetomium thermophilum</i> FDH in <i>Pichia pastoris</i>

Nowadays, the use of formate dehydrogenase (FDH, EC 1.17.1.9) is well established as a means of NADH regeneration from NAD+ via the coupled conversion of formate into carbon dioxide. Recent studies have been reported that specifically Chaetomium thermophilum FDH (CtFDH) is the most efficient FDH catalyzing this reaction in reverse (i.e. using CO2 as a substrate to produce formate, and thereby regenerating NAD+). However, to date the production of active CtFDH at high protein expression levels has received relatively little attention. In this study, we have tested the effect of batch and high cell density fermentation (HCDF) strategies in a small stirred fermenter, as well as the effect of supplementing the medium with casamino acids, on the expressed level of secreted CtFDH using P. pastoris. We have established that the amount of expressed CtFDH was indeed enhanced via a HCDF strategy and that extracellular protease activity was eliminated via the addition of casamino acids into the fermentation medium. On this basis, secreted CtFDH in an active form can be easily separated from the fermentation and can be used for subsequent biotechnological applications. © 2020 Elsevier Inc.Düzce ÃœniversitesiThis work was supported by Düzce University Scientific Research Project Department with 2017.06.02.578 project number and special thanks to BERC Lab for providing p PICZ? A vector with 6xHis-tagged Ct FDH. The authors also gratefully acknowledge the help and support of Rowan M Lindeque (Department of Chemical and Biochemical Engineering, DTU, Denmark)

Vaccination status of COVID-19 patients followed up in the ICU in a country with heterologous vaccination policy: A multicenter national study in Turkey

Objective: Vaccination against severe acute respiratory syndrome coronavirus-2 (SARS-2) prevents the development of serious diseases has been shown in many studies. However, the effect of vaccination on outcomes in COVID-19 patients requiring intensive care is not clear. Methods: This is a retrospective multicenter study conducted in 17 intensive care unit (ICU) in Turkey between January 1, 2021, and December 31, 2021. Patients aged 18 years and older who were diagnosed with COVID-19 and followed in ICU were included in the study. Patients who have never been vaccinated and patients who have been vaccinated with a single dose were considered unvaccinated. Logistic regression models were fit for the two outcomes (28-day mortality and in-hospital mortality). Results: A total of 2968 patients were included final analysis. The most of patients followed in the ICU during the study period were unvaccinated (58.5%). Vaccinated patients were older, had higher Charlson comorbidity index (CCI), and had higher APACHE-2 scores than unvaccinated patients. Risk for 28-day mortality and in-hospital mortality was similar in across the year both vaccinated and unvaccinated patients. However, risk for in-hospital mortality and 28-day mortality was higher in the unvaccinated patients in quarter 4 adjusted for gender and CCI (OR: 1.45, 95% CI: 1.06–1.99 and OR: 1.42, 95% CI: 1.03–1.96, respectively) compared to the vaccinated group. Conclusion: Despite effective vaccination, fully vaccinated patients may be admitted to ICU because of disease severity. Unvaccinated patients were younger and had fewer comorbid conditions. Unvaccinated patients have an increased risk of 28-day mortality when adjusted for gender and CCI