آنالیز ساختاری آنزیم آسپارتات کیناز در کورینه باکتریوم به منظور پیشنهاد بهترین ساختار آنزیم جهت افزایش تولید صنعتی اسید آمینه لیزین

نوع مقاله : علمی پژوهشی- ژنتیک و اصلاح دام و طیور

نویسندگان

1 گروه علوم دامی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

2 گروه علوم دامی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران.

3 گروه میکروبیولوژی و ایمونولوژی، دانشکده دامپزشکی، دانشگاه تهران

چکیده

آنزیم آسپارتات کیناز یکی از آنزیم‌های کلیدی و حساس در متابولیسم تولید لیزین در طیف وسیعی از جانوران بخصوص گونه‌های مختلف میکروارگانیسم کورینه باکتریوم می‌باشد. هدف از این تحقیق بررسی بیوانفورماتیکی آنزیم آسپارتات کیناز در گونه‌های مختلف کورینه باکتریوم توسط پایگاه های معتبر می‌باشد تا بهترین ساختار بیوانفورماتیکی آنزیم آسپارتات کیناز به منظور استفاده برای تولید لیزین در مقیاس صنعتی پیشنهاد شود.  ویژگی های تکاملی، فیزیولوژیکی و فیزیکوشیمیایی آنزیم آسپارتات کیناز در 33 گونه مختلف کورینه باکتریوم توسط پایگاه ها و نرم افزارهای MEGA 6.6، ProtScale و ProtParam مورد بررسی قرار گرفت.  سپس ساختار سه بعدی پروتئین‌های مذکور به روش هومولوژی و با استفاده از سرور Swiss-Model مدل سازی شد و ارزیابی کیفیت مدل های بدست آمده پس از بهینه سازی انرژی با استفاده از ابزار Rampage صورت گرفت. نتایج تجزیه و تحلیل درخت تکاملی نشان دادند که زمان اشتقاق پروتئین آسپارتات کیناز در 33 گونه کورینه باکتریوم مورد مطالعه، بسیار بهم نزدیک می‌باشد. نتایج بررسی پایگاه های ProtScale و ProtParam نشان داد که آنزیم آسپارتات کیناز در گونه کورینه باکتریوم گلوتامیکوم با شماره دستیابی CAO00530.1 و SJM57548.1 دارای بهترین خواص فیزیکوشیمیایی و بیشترین پایداری می‌باشند. سپس با بررسی های بیشتر توسط سرور Swiss-Model و ابزار Rampage مشخص شد که دو پروتیئن CAO00530.1 و SJM57548.1 دارای بهترین ساختار سه بعدی می‌باشند. از نتایج آنالیز in silico به دست آمده می‌توان چنین استنباط نمود که احتمالا آنزیم آسپارتات کیناز با شماره های دستیابی بالا دارای بهترین خواص فیزیکو شیمیایی، بیشترین پایداری و همچنین بهترین ساختار سه بعدی نسبت به دیگر پروتئین‌های مورد بررسی در این مطالعه بود. این یافته ها می تواند برای بهترین نوع آنزیم آسپارتات کیناز به منظور تولید نوترکیب در شرایط آزمایشگاهی مفید باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Structural analysis of aspartate kinase enzyme in Corynebacterium species to find the best enzyme structure for high industrial production of lysine amino acid

نویسندگان [English]

  • Morteza Hashemi َAttar 1
  • Mohammad Reza Nassiry 1
  • Ali Javadmanesh 2
  • Bahar Nayeri Fasaei 3
1 Department Animal Science, Faculty of agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
2 Department Animal Science, Faculty of agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
3 Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Iran
چکیده [English]

Introduction Lysine is one of the essential amino acids not synthesized biologically in the human body and mammals so should be supplied through diets. Among the industrially important amino acids, L-lysine is in 1st position, which is used in pharmaceuticals, animals, human feeds and precursors for the production of peptides or agrochemicals. As L-lysine has large applications, the demand for it is increasing constantly year by year. To minimize the gap between increasing demand and production of L-lysine, it has to be produced in large scale. Corynebacterium species especially Corynebacterium glutamicum is widely used for the industrial production of amino acids especially L-glutamate and L- lysine. The C. glutamicum from long period has been used for the industrial production of various amino acids, primary metabolites and nucleotides. This organism is an aerobic gram positive, rod shaped and non-sporulating bacteria which used for the industrial production of amino acids of L-lysine and L-glutamate. This bacterium uses a variety of carbohydrates, alcohols and organic acids as single sources of carbon and energy for growth and also for the amino acid production. The quantity of lysine production by wild (natural) type of Corynebacterium glutamycum is very low, and its cultivation and propagation cannot provide the amino acid required by markets, therefor the wild type of this bacterium is not suitable and cost-effective for industrial purposes. To minimize the gap between increasing demand and production of L-lysine, it has to be produced in large scale. Aspartate kinase (AK) is one of the key enzymes in the biosynthesis of aspartate-derived amino acids such as lysine. This enzyme catalyzes the transfer of the C-phosphate group of ATP to aspartic acid. In most bacteria, the reaction is the first step of branched biosynthetic pathway for lysine, threonine, isoleucine and methionine and is known to be regulated by the end metabolites through feedback inhibition. For example, aspartate kinase from Corynebacterium glutamicum is concertedly inhibited by lysine and threonine, while aspartate kinase I and III from Escherichia coli is inhibited by threonine and lysine, respectively. Due to industrial production of lysine amino acid by using Corynebacterium species, a lot of researches have been done to improve the genetic modification of these microorganisms. Today, bioinformatics tools are available as online access through web-based databases and software, which can be used to study best structures of aspartate kinase enzyme with the least cost and time.  Also due to high laboratory costs, the use of bioinformatics methods will be important in obtaining the final result. The aim of this study was to investigate the bioinformatics structure of aspartate kinase enzyme in different species of Corynebacterium by authenticated bioinformatics databases to suggest best bioinformatics structure of the aspartate kinase enzyme for applying in laboratory cloning and production of lysine amino acid for industrial purposes.
Material and methods The amino acid sequences of the aspartic kinase enzyme from 33 species of the Corynebacterium was obtained from the NCBI (https://www.ncbi.nlm.nih.gov/protein) and stored as FASTA. In order to study the genetic distances and similarities in 33 species of Corynebacterium a phylogenetic tree was constructed using the Neighbor Joining method using the Mega software (MEGA 6). (A bootstrap check with 1000 replications was also conducted to obtain a confidence level for the branches) ClC main work bench5 software was used to investigate genetic similarities using protein sequences. The Evolutionary properties, physiological and physicochemical properties of aspartate kinase enzyme were studied and investigated in 33 different species of Corynebacterium through valid databases and software of NCBI, MEGA, ProtScale and ProtParam. In order to predict the second structure, two proteins selected from the psipred server were used (http://bioinf.cs.ucl.ac.uk/psipred). For this purpose, the protein sequences of aspartate kinase enzyme in Corynebacterium glutamicum with access numbers of CAO00530.1 and SJM57548.1 introduced into the psipred and their second structure was mapped. Afterward, three-dimensional structure of mentioned protein was modeled using Swiss-model server (https://swissmodel.expasy.org) Then the quality of the two predicted models evaluated by the Rampage server (http://mordred.bioc.cam.ac.uk/ ~ rapper / rampage2.php), and in the next step its ramachandran plot mapped.
Results and discussions The results of evolutionary tree analysis in Corynebacterium species showed that derivation time of aspartate kinase protein in these 33 species is very close. The results of the ProtScale and ProtParam databases showed that the aspartatekinase enzyme of Corynebacterium glutamicum with the access number of CAO00530.1 and SJM57548.1 have the best physicochemical and maximum stability among 33 different species of study. Afterward, with further in silico investigation by the Swiss-Model server and Rampage tool, it was found that the two access numbers of CAO00530.1 and SJM57548.1 had the best three-dimensional structure. From the results of in silico analyses, it can be inferred that the aspartate kinase enzyme with the two access numbers of CAO00530.1 and SJM57548.1 have the best physicochemical properties, the most stable and also the best three-dimensional structure and therefore could be offered for laboratory cloning and production of lysine amino acid for industrial purposes.
Conclusion Due to wide applications and importance of lysine production in our country and also the necessity of selecting appropriate strain of Corynebacterium for genetic engineering and industrial production, this bioinformatics study was done to predict best structure of aspartate kinase enzyme and best strain of Corynebacterium. Based on the results of our in silico analysis, it is suggested that corynebacterium glutamicum has the best protein structure of aspartate kinase enzyme and may be beneficial to increase Industrial lysine amino acid production.

کلیدواژه‌ها [English]

  • Aspartate kinase
  • Bioinformatics
  • Corynebacterium
  • Lysine amino acid production
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