Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/16166
Title: Molecular Characterization of Novel Fiber Degrading Enzymes from Microbial Sources and their Application in Animal Feed
Authors: Raza, Ahmad
Keywords: Biological & Medical Sciences
Biotechnology
Issue Date: 2021
Publisher: Pakistan Institute of Engineering & Applied Sciences, Islamabad.
Abstract: Cellulases (exo-glucanase, endo-glucanase and β-glucosidase) and xylanases are among the most important fiber degrading enzymes. These enzymes have engrossed considerable attention due to their vast applications in various industries. Animal feed industry is one of the essential industries for the application of these enzymes. Present study was aimed to isolate microbes with the potential to produce these fiber degrading enzymes, their molecular characterization along with their application in animal feed. In this study we isolated 101 bacteria from buffalo dung and rumen samples and subjected to plate based screening for fiber degrading potential. From all isolated bacteria, twenty two were screened for fiber degradation potential and subjected to BLAST algorithm and identified as members of 2 major phyla including firmicutes and actinobacteria with 3 families as bacillaceae, enterococcaceae and streptomycetaceae. The enzyme activity index showed, four (4) best enzyme producers including BR28, BR96 with highest xylanase activity, BD69 with highest β-glucosidase activity and BD92 with highest endo-glucanase and exo-glucanase activities. The potential isolate BD92 (Bacillus sonorensis) was chosen for statistical optimization with response surface methodology using central composite design. The maximum exo-glucanase, endo-glucanase, β-glucosidase and xylanase activities were achieved under optimized conditions (carboxymethyl cellulose 2%, yeast extract 1.5% and time 72 h) were 671.68, 1460.00, 361.29 and 5690.00 U L-1 , respectively. Similarly, the highest co-production of multi-enzyme cocktail BsBD92 (exo-glucanase, endo-glucanase, β-glucosidase and xylanase) was achieved on wheat bran (WB) followed by wheat straw, cotton stalk and rice straw when used as cheap alternative carbon sources. Maximum in vitro enzymatic saccharification was observed for WB after 24 h using multi-enzyme cocktail BsBD92. All enzymes showed more than 20% residual activates at highest temperature (90 °C) and > 70% activities at pH 5.5 and 50 °C. β-glucosidase (Bteqβgluc) from B. tequilensis BD69 and endo-glucanase (BsEgl) from B. sonorensis BD92 were also cloned for molecular characterization. The Bteqβgluc gene encodes a protein of 433 amino acids (AA) and belong to glycoside hydrolase 4 (GH4) family. Pichia pastoris was better expression system with xviii extracellular secretion of Bteqβgluc protein and high enzymatic activity. As Bteqβgluc retains > 50% of its activity at 80 °C and > 80% activity at optimum pH after 60 min. However, BsEgl gene encodes a protein of 499 AA and belongs to GH5 family with cellulose binding domain from superfamily 3. BsEgl was stable at a range of pH from 4-8 for 60 min and 50 °C for 180 min. Finally, the application of multi-enzyme cocktail BsBD92 and recombinant enzymes was tested for improvement of growth performance in broiler birds. Multi enzyme cocktail BsBD92 gave better results in term of production index and feed conversion ratio. While, combination of recombinant enzymes (Bteqβgluc and BsEgl) also showed better performance than single enzymes.
Gov't Doc #: 22858
URI: http://prr.hec.gov.pk/jspui/handle/123456789/16166
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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