Recombinant Expression in <i>Bacillus megaterium</i> and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 <i>α</i>-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome
There is no doubt that ruminants have the capability to digest lignocellulosic compounds and to utilize them as an absorbable form of energy by tapping into enzymes produced by the microbial population in their rumens. Among the rumens of various ruminants, this study focused on Korean goat rumens b...
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MDPI AG
2023-10-01
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| Series: | Applied Microbiology |
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| Online Access: | https://www.mdpi.com/2673-8007/3/4/80 |
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| author | Sazzad Hossen Toushik Md. Ashrafudoulla |
| author_facet | Sazzad Hossen Toushik Md. Ashrafudoulla |
| author_sort | Sazzad Hossen Toushik |
| collection | DOAJ |
| description | There is no doubt that ruminants have the capability to digest lignocellulosic compounds and to utilize them as an absorbable form of energy by tapping into enzymes produced by the microbial population in their rumens. Among the rumens of various ruminants, this study focused on Korean goat rumens because of their unique digestibility of lignocellulosic biomasses. Therefore, a novel <i>Gene12</i> gene was screened and unmasked from the constructed rumen metagenomic library of a Korean black goat and expressed in a <i>Bacillus megaterium</i> system. The recombinant protein was distinguished as a novel <i>α</i>-L-arabinofuranosidase enzyme from glycosyl hydrolase family 43 (GH43) for its capability to hydrolyze the non-reducing end of <i>α</i>-1,5-L-arabinofuranose linkages in <i>α</i>-L-arabinofuranosyl groups. The enzyme can also break apart <i>α</i>-L-arabinofuranosidic linkages and act synergistically with other hemicellulolytic enzymes to release <i>α</i>-1,2- and <i>α</i>-1,3-L-arabinofuranosyl groups from L-arabinose-comprising polysaccharides. In silico, phylogenetic, and computational analyses proclaimed that the <i>Gene12</i> gene encodes a novel carbohydrate-active enzyme possessing a V-shaped indentation of the GH43 catalytic and functional domain (carbohydrate-binding module 6). The recombinant Gene12 protein has shared 81% sequence homology with other members of the GH43 family. Enzymic synopses (optimal pH, temperatures, and stability studies) of the recombinant Gene12 enzyme and its substrate specificity (synthetic and natural substrates) profiling were considered. The recombinant Gene12 <i>α</i>-L-arabinofuranosidase works best at pH 6.0 and 40 °C, and it is stable at pH 4.0 to 7.0 at temperatures of 20 to 50 °C. Additionally, 5-blended β-sheets were identified through a tertiary (3D) structure analysis along with the high substrate specificity against <i>p</i>-nitrophenyl-D-arabinofuranoside (<i>p</i>NPA). The highest substrate specificity of <i>p</i>NPA for Gene12 <i>α</i>-L-arabinofuranosidase indicated its confirmation as an exo-type arabinofuronidase. The results thus propose using the Gene12 protein as an exo-mannered GH43 <i>α</i>-L-arabinofuranosidase (EC 3.2.1.55) enzyme. |
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| spelling | doaj.art-5414842af5b24e0dbe1270658053abc82023-12-29T15:46:05ZengMDPI AGApplied Microbiology2673-80072023-10-01341164117710.3390/applmicrobiol3040080Recombinant Expression in <i>Bacillus megaterium</i> and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 <i>α</i>-L-Arabinofuranosidase from the Korean Black Goat Rumen MetagenomeSazzad Hossen Toushik0Md. Ashrafudoulla1Department of Biochemistry and Microbiology, School of Health and Life Sciences, North South University, Dhaka 1229, BangladeshDepartment of Food Science and Technology, Chung-Ang University, Anseong 456-756, Republic of KoreaThere is no doubt that ruminants have the capability to digest lignocellulosic compounds and to utilize them as an absorbable form of energy by tapping into enzymes produced by the microbial population in their rumens. Among the rumens of various ruminants, this study focused on Korean goat rumens because of their unique digestibility of lignocellulosic biomasses. Therefore, a novel <i>Gene12</i> gene was screened and unmasked from the constructed rumen metagenomic library of a Korean black goat and expressed in a <i>Bacillus megaterium</i> system. The recombinant protein was distinguished as a novel <i>α</i>-L-arabinofuranosidase enzyme from glycosyl hydrolase family 43 (GH43) for its capability to hydrolyze the non-reducing end of <i>α</i>-1,5-L-arabinofuranose linkages in <i>α</i>-L-arabinofuranosyl groups. The enzyme can also break apart <i>α</i>-L-arabinofuranosidic linkages and act synergistically with other hemicellulolytic enzymes to release <i>α</i>-1,2- and <i>α</i>-1,3-L-arabinofuranosyl groups from L-arabinose-comprising polysaccharides. In silico, phylogenetic, and computational analyses proclaimed that the <i>Gene12</i> gene encodes a novel carbohydrate-active enzyme possessing a V-shaped indentation of the GH43 catalytic and functional domain (carbohydrate-binding module 6). The recombinant Gene12 protein has shared 81% sequence homology with other members of the GH43 family. Enzymic synopses (optimal pH, temperatures, and stability studies) of the recombinant Gene12 enzyme and its substrate specificity (synthetic and natural substrates) profiling were considered. The recombinant Gene12 <i>α</i>-L-arabinofuranosidase works best at pH 6.0 and 40 °C, and it is stable at pH 4.0 to 7.0 at temperatures of 20 to 50 °C. Additionally, 5-blended β-sheets were identified through a tertiary (3D) structure analysis along with the high substrate specificity against <i>p</i>-nitrophenyl-D-arabinofuranoside (<i>p</i>NPA). The highest substrate specificity of <i>p</i>NPA for Gene12 <i>α</i>-L-arabinofuranosidase indicated its confirmation as an exo-type arabinofuronidase. The results thus propose using the Gene12 protein as an exo-mannered GH43 <i>α</i>-L-arabinofuranosidase (EC 3.2.1.55) enzyme.https://www.mdpi.com/2673-8007/3/4/80rumenmetagenome<i>α</i>-L-arabinofuranosidase<i>Bacillus megaterium</i>glycosyl hydrolase family 43 |
| spellingShingle | Sazzad Hossen Toushik Md. Ashrafudoulla Recombinant Expression in <i>Bacillus megaterium</i> and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 <i>α</i>-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome Applied Microbiology rumen metagenome <i>α</i>-L-arabinofuranosidase <i>Bacillus megaterium</i> glycosyl hydrolase family 43 |
| title | Recombinant Expression in <i>Bacillus megaterium</i> and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 <i>α</i>-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome |
| title_full | Recombinant Expression in <i>Bacillus megaterium</i> and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 <i>α</i>-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome |
| title_fullStr | Recombinant Expression in <i>Bacillus megaterium</i> and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 <i>α</i>-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome |
| title_full_unstemmed | Recombinant Expression in <i>Bacillus megaterium</i> and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 <i>α</i>-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome |
| title_short | Recombinant Expression in <i>Bacillus megaterium</i> and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 <i>α</i>-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome |
| title_sort | recombinant expression in i bacillus megaterium i and biochemical characterization of exo mannered glycosyl hydrolase family 43 i α i l arabinofuranosidase from the korean black goat rumen metagenome |
| topic | rumen metagenome <i>α</i>-L-arabinofuranosidase <i>Bacillus megaterium</i> glycosyl hydrolase family 43 |
| url | https://www.mdpi.com/2673-8007/3/4/80 |
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