Cloning and expression of recombinant endo 1,4 β xylanase in origami strain and evaluation of microbial enzyme supplementation effects on broiler performance

Document Type : Research Paper


1 Department of Biotechnology, Faculty of Agriculture, University of Shahrekord, Chaharmahal and baktyari province, IRAN

2 PhD Student of Biotechnology, Department of Biotechnology, Institute of Science, High Technology and Environmental Sciences, University of Advanced Technology, Kerman, Iran.

3 Student of MSc in Biotechnology, Faculty of Agriculture, Shahrekord University


Today, one of the ways to reduce production costs in poultry industrial units is to replace cost-effective grains such as wheat with corn in the poultry diet. However, the presence of non-starch polysaccharides in the cell wall of wheat has inevitably limited the uptake of nutrients by poultry. In this regard, one of the methods to remove non-starch polysaccharides is the use of xylanase enzyme additive in formulated diets based on wheat. For this purpose, the aim of this experiment was to clone the recombinant enzyme xylanase and investigate its effects on the performance of wheat-fed broiler chickens.
Materials and methods
In current investigation, in the first step, xylanase gene, as a degradation enzyme of non-starch polysaccharides in wheat-based poultry diet, was cloned into plasmid pET-21a (+) in E. coli origami expression strain using heat shock method. IPTG inducer was used to induce gene expression and the expression result was observed on SDS-PAGE. Then, in the second step, the recombinant xylanase enzyme solution was added to the diet of one-hundred Ross 308 broilers in a completely randomized design. Then, the mean body weight gain (BWG), feed conversion ratio (FCR) and viscosity of jejunum contents were also measured.
The results of present study showed that the recombinant xylanase gene was transferred to origami and the cloning results of PCR confirmed the presence of a specific band of 966 bp. SDS-PAGE also showed a recombinant protein with a molecular weight of 35 kDa. Furthermore, the results of adding recombinant xylanase enzyme to wheat-based diet showed an increase in the weight of broilers and a decrease in feed conversion ratio and viscosity in the final period and the whole period compared to the control group at a probability level of 5%.
The results of the current research showed that the production of recombinant enzyme xylanase and its addition as a dietary supplement in wheat-based diets could increase the yield of broilers by breaking down arabinosylans in the wheat cell wall and the presence of xylanase enzyme in the diet is known as one of the important factors in enhancing the growth of broilers.


احسنی محمدرضا، محمدآبادی محمدرضا، اسدی فوزی و همکاران (1398) بیان ژن لپتین در بافت چربی زیرپوستی گاوهای هلشتاین با استفاده از Real Time PCR. مجله بیوتکنولوژی کشاورزی 11(1)، 150-135.
توحیدی نژاد فاطمه، محمدآبادی محمدرضا، اسمعیلی زاده کشکوئیه علی، نجمی نوری عذرا (1393) مقایسه سطوح مختلف بیان ژنRheb  در بافت های مختلف بز کرکی راینی. مجله بیوتکنولوژی کشاورزی 6(4)، 50-35.
جعفری دره در امیر حسین، محمدآبادی محمدرضا، اسمعیلی زاده کشکوئیه علی، ریاحی مدوار علی (1395) بررسی بیان ژن CIB4  در بافت­های مختلف گوسفند کرمانی با استفاده از Real Time qPCR. مجله پژوهش در نشخوارکنندگان 4(4)، 132-119.
محمدآبادی محمدرضا (1399) بیان ژن ESR1 در بز کرکی راینی با استفاده از Real Time PCR‎. مجله بیوتکنولوژی کشاورزی 12(1)، 192-177.
محمدآبادی محمدرضا (1399) پروفایل بیانی mRNA مختص بافت ژن ESR2 در بز. مجله بیوتکنولوژی کشاورزی 12(4)، 181-167.
محمدی فر آمنه، فقیه ایمانی سید علی، محمدآبادی محمد رضا، سفلایی محمد (1392) تأثیر ژن TGFb3 بر ارزش های فنوتیپی و ارثی صفات وزن بدن در مرغ بومی استان فارس. مجله بیو تکنولوژی کشاورزی  5(4)، 136-125.
Abdollahi MR, Ravindran V, Wester TJ et al. (2018) Influence of feed form and conditioning temperature on performance, apparent metabolisable energy and ileal digestibility of starch and nitrogen in broiler starters fed wheat-based diet. Anim Feed Sci Tech 168, 88-99.
Ahsani MR, Mohammadabadi MR, Asadi Fozi M et al. (2019a) Effect of Roasted Soybean and Canola Seeds on Peroxisome Proliferator‐Activated Receptors Gamma (PPARG) Gene Expression and Cattle Milk Characteristics. Iran J Appl Anim Sci 9, 635-642.
Ahsani MR, Mohammadabadi MR, Asadi Fozi M et al. (2019b) Leptin gene expression in subcutaneous adipose tissue of Holstein dairy cattle using Real Time PCR. Agric Biotechnol J 11, 135-150 (In Persian).
Bhardwaj N, Kumar B, Verma P et al. (2019) A detailed overview of xylanases: an emerging biomolecule for current and future prospective. Bioresour Bioprocess 6, 40.
Bilgin S, Ulusu Y, Kudus H et al. (2018) Cloning, Expression and characterization of xylanase (xyn-akky1) from Bacillus subtilis in Escherichia coli. Sakarya Uni J Sci 6, 1508-1517.
Brenes A, Smith M, Guenter W et al. (2019) Effect of enzyme supplementation on the performance and digestive tract size of broiler chickens fed wheat- and barley based diets. Poultry Science J Nut 125, 947-955.
Buchanan NP, Kimbler LB, Parsons AS et al. (2007) The effects of non-starch polysaccharide enzyme addition and dietary energy restriction on performance and carcass quality of organic broiler chickens. J App Poult Res 10, 1-12.
Burkholder KM, Thompson KL, Einstein ME et al. (2011) Influence of stressors on normal intestinal microbiota, intestinal morphology, and susceptibility to Salmonella Enteritidis colonization in broilers. Poult Sci 87, 1734-1741.
Cao G, Zhang X, Zhong L et al. (2011) A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides. Biotechnol Lett 33, 1047-1051.
Fajardo P, Pastrana L, Endez J (2012) Effects of feeding of two potentially probiotic preparations from lactic acid on the performance and fecal microflora of broiler chickens. Sci World J 23, 1-9.
Gao F, Jiang Y, Zhou GH et al. (2008) The effects of xylanase supplementation on performance, characteristics of the gastrointestinal tract, blood parameters and gut microflora in broilers fed on wheat-based diets. Anim Feed Sci Tech 142, 173-184.
Ghorbani MR, Fayazi J, Chaji M (2019) Effect of dietary phytase and NSP-degrading enzymes in diets containing rapeseed meal on broiler performance and carcass characteristics. Res J Biological Sci 4, 258-264.
Hong D, Adeola O (2018) Addition of enzyme to starter and grower diets for ducks. Poult Sci 81, 1842-1849.
Jha R, Das R, Oak S et al. (2020) Probiotics (direct-fed microbial) in poultry nutrition and their effects on nutrient utilization, growth and laying performance, and gut health: A systematic review. Animals 10, 1-18.
Junqueira L, Carneiro J, Kelly O et al. (2020) Basic Histology, 8th Ed, Editora Guanabara kogan S.A. pp. 289-296.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Liu C (2013) Strategies for designing transgenic DNA constructs. Methods Molecular Biology 1027, 1-16.
Masoudzadeh SH, Mohammadabadi M, Khezri A, et al. (2020) Effects of diets with different levels of fennel (Foeniculum vulgare) seed powder on DLK1 gene expression in brain, adipose tissue, femur muscle and rumen of Kermani lambs. Small Rumin Res 193, e106276.
Moazeni S, Mohammadabadi MR, Sadeghi M (2010) Association between UCP gene polymorphisms and growth, breeding value of growth and reproductive traits in mazandaran indigenous chicken. Open J. Anim. Sci 6, 1-8.
Mohammadabadi MR (2021) Tissue-specific mRNA expression profile of ESR2 gene in goat. Agric Biotechnol J 12 (4), 167-181 (In Persian).
Mohammadabadi MR (2020) Expression of ESR1 gene in Raini Cashmere goat using Real Time PCR. Agric Biotechnol J 12 (1), 177-192 (In Persian).
Mohammadabadi MR, Kord M, Nazari M (2018) Studying expression of leptin gene in different tissues of Kermani Sheep using Real Time PCR. Agric Biotechnol J 10, 111-122 (in Persian).
Mohammadabadi MR, Tohidinejad F (2017) Charachteristics determination of Rheb gene and protein in Raini Cashmere goat. Iran J Appl Anim Sci 7, 289-295.
Mohammadifar A, Mohammadabadi MR (2017) The effect of uncoupling protein polymorphisms on growth, breeding value of growth and reproductive traits in the fars indigenous chicken. Iran J Appl Anim Sci 7, 679-685.
Mohammadifar A, Mohammadabadi MR (2018) Melanocortin-3 receptor (mc3r) gene association with growth and egg production traits in fars indigenous chicken. Malays Appl Biol 47, 85–90.
Mohammadifar A, Faghih Imani SA, Mohammadabadi MR (2014) The effect of TGFb3 gene on phenotypic and breeding values of body weight traits in Fars native fowls. J. Agric. Biotech 5, 125-136 (In Persian).
Sadeghi Alikelayeh A, Mirakhorli N, Akbari MR, Shareghi Brojeni B (2020) Cloning and expression of recombinant xylanase enzyme (xynA) in E. coli. Agric Biotechnol J 4, 1-22.
Serranom M, Frikha PM, Corchero J (2020) Influence of feed form and source of soybean meal on growth performance, nutrient retention, and digestive organ size of broilers, 2. Battery Poult Sci 92, 693-708.
Tohidi nezhad F, Mohammadabadi MR, Esmailizadeh AK, Najmi Noori A (2015) Comparison of different levels of Rheb gene expression in different tissues of Raini Cashmir goat. Agric Biotechnol J 6, 35-50.
Vandeplas S, Dauphin RD, Thonart P et al. (2010) Effect of the bacterial or fungal origin of exogenous xylanases supplemented to a wheat-based diet on performance of broiler chickens and nutrient digestibility of the diet. Can J Anim Sci 90, 221-228.
Wang J, Zhang H, Wu M (2011) Cloning and sequence analysis of a novel xylanase gene, Auxyn10A, from Aspergillus usamii. Biotechnol Lett 33, 1029-1038.
Wang W, Yan-long J, Yi-chun L et al. (2018) Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells. Sci Rep 7, 10416.
Zafar A, Aftab MN, Din ZU et al. (2016) Cloning, expression and purification of xylanase gene from Bacillus licheniformis for use in saccharification of plant biomass. Appl Biochem Biotechnol 178, 294–311.