Publication: Tyrosinase-based melanin production in corynebacterium glutamicum
Abstract
Tahmini olarak 30 milyar doları aşan değeri ile pigmentler, dünya çapında büyük bir ekonomik öneme sahiptir. Bu pigmentlerden biri olan melanin, kimyasal yapısına göre 4 farklı kategoriye ayrılır: eumelanin, feomelanin, nöromelanin ve allomelanin. Eumelanin, siyah ila kahverengi tonlarda ve tirozinin L-3,4-dihidroksifenilalanine (L-DOPA) oksidatif polimerizasyon türevleri ile oluşturulan melanin bir alt grubu olarak kabul edilir. Eumelanin üreten kaynaklardan biri, öncüleri tirozin veya L-DOPA olan bakterilerdir. Bu tezde ekonomik melanin pigmentinin üretimi için üç farklı Corynebacterium glutamicum hücresinde üretim araştırılmıştır. İlk olarak, Cg-EKV-I hücresi kullanılarak tirozinaz tarafından besi yerine ilave edilen tirozin L-DOPA'ya dönüştürülmüş ve sonrasında L-DOPA dopakinon'a dönüşmüştür. Dopakinon kendiliğinden okside olmuş ve melanin polimerleri oluşmuştur. Önce bu hücre ile melaninin optimum eldesi, üretim şişesi şekli, üretim hacmi ve rotasyon hızı incelenerek belirlenmiştir. Ek olarak, üretime başladıktan 24 saat sonra, toz L-tirozin eklenerek ilave edilen tirozinin üretime ne kadar katkı sağladığı incelenmiştir. Elde edilen deneysel sonuçlar tirozin üreticisi AROM3D hücresiyle üretim için kullanılmıştır. Modelleme temelli optimizasyon ile AROM3D hücrelerinin maksimum melanin üretim koşulları belirlenmiştir. Tirozin ilavesi ile Cg-EKV-I hücreleri elde edilen maksimum melanin üretimi 0.75 g/ L iken, tirozin ilavesi olmadan AROM3D hücresinden optimizasyon sonrasında elde edilen melanin üretimi 1.03 g/ L olmuştur. Optimizasyon sonrasında elde edilen melanin saflaştırılmıştır. Üretilen bu pigmentin melanin olduğu HPLC, UV-Vis, FT/ IR ve SEM analizleri ile gösterilmiştir. Son olarak, Cg-EKV-II hücresi ile portakal kabuğu hidrolizi kullanılarak melanin üretimi gerçekleştirilmiştir. Hücre miktarı, sıcaklık ve rotasyon hızı optimize edilmiştir. Bu tez çalışmasıyla, melanin pigmentinin optimum ve ekonomik olarak uygun bir şekilde elde edilmesi sağlanmıştır.
Pigments, with an estimated value surpassing 30 billion dollars in the industry, hold significant economic importance worldwide. Melanin, one of these pigments, is categorized into four distinct types based on its chemical structure: eumelanin, pheomelanin, neuromelanin, and allomelanin. Eumelanin, ranging from black to brown tones, is considered a subcategory of melanin formed by oxidative polymerization derivatives of tyrosine such as L-3,4-dihydroxyphenylalanine (L-DOPA). One of the sources producing eumelanin is bacteria with precursors like tyrosine or L-DOPA. This thesis investigates the production of economical melanin pigment using three different Corynebacterium glutamicum cells. Initially, using the Cg-EKV-I cell, tyrosine added instead of a growth medium is converted to L-DOPA by tyrosinase and subsequently L-DOPA transforms into dopaquinone. Dopaquinone undergoes auto-oxidation, resulting in the formation of melanin polymers. The optimal conditions for melanin production with this cell, including production vessel shape, volume, and rotation speed, were determined. Additionally, the contribution of added tyrosine to production was examined by adding powdered L-tyrosine 24 hours after the start of production. The experimental results were used for production with the tyrosine-producing AROM3D cell. Using modeling-based optimization, the maximum melanin production conditions for AROM3D cells were determined. While the maximum melanin production from Cg-EKV-I cells with tyrosine addition was 0.75 g/ L, the melanin production after optimization from AROM3D cells without tyrosine addition was 1.03 g/ L. The obtained melanin was purified after optimization. The produced pigment's identification as melanin was confirmed through HPLC, UV-Vis, FT/ IR, and SEM analyses. Lastly, melanin production was attempted using orange peel hydrolysate with the Cg-EKV-II cell, with optimization of cell amount, temperature, and rotation speed. This thesis ensures the optimum and economically feasible production of melanin pigment.
Pigments, with an estimated value surpassing 30 billion dollars in the industry, hold significant economic importance worldwide. Melanin, one of these pigments, is categorized into four distinct types based on its chemical structure: eumelanin, pheomelanin, neuromelanin, and allomelanin. Eumelanin, ranging from black to brown tones, is considered a subcategory of melanin formed by oxidative polymerization derivatives of tyrosine such as L-3,4-dihydroxyphenylalanine (L-DOPA). One of the sources producing eumelanin is bacteria with precursors like tyrosine or L-DOPA. This thesis investigates the production of economical melanin pigment using three different Corynebacterium glutamicum cells. Initially, using the Cg-EKV-I cell, tyrosine added instead of a growth medium is converted to L-DOPA by tyrosinase and subsequently L-DOPA transforms into dopaquinone. Dopaquinone undergoes auto-oxidation, resulting in the formation of melanin polymers. The optimal conditions for melanin production with this cell, including production vessel shape, volume, and rotation speed, were determined. Additionally, the contribution of added tyrosine to production was examined by adding powdered L-tyrosine 24 hours after the start of production. The experimental results were used for production with the tyrosine-producing AROM3D cell. Using modeling-based optimization, the maximum melanin production conditions for AROM3D cells were determined. While the maximum melanin production from Cg-EKV-I cells with tyrosine addition was 0.75 g/ L, the melanin production after optimization from AROM3D cells without tyrosine addition was 1.03 g/ L. The obtained melanin was purified after optimization. The produced pigment's identification as melanin was confirmed through HPLC, UV-Vis, FT/ IR, and SEM analyses. Lastly, melanin production was attempted using orange peel hydrolysate with the Cg-EKV-II cell, with optimization of cell amount, temperature, and rotation speed. This thesis ensures the optimum and economically feasible production of melanin pigment.