Expression, characterization, and optimization of recombinant glycoside hydrolases from rare marine bacteria Jeotgalibacillus Malaysiensis D5T sp.nov.

Starch-based sugars serve as raw materials in food industry. These sugars are produced using liquefaction and saccharification processes. These methods require hightemperature gelatinization of starch granules during liquefaction, followed by enzymatic saccharification using several glycoside hydrolases (GHs) to obtain sugar syrups. However, starch-based products are costly due to high energy consumption during the conversion process. Raw starch-degrading enzymes able to directly degrade raw starch granules would simplify the entire starch degradation process and allow energy savings. For this reason, this study investigated three GHs from a rare marine bacterium Jeotgalibacillus malaysiensis D5T. During the first stage of this study, the GHs information from the genome of strain D5T was mined. α-amylase (AmyJM), type I pullulanase (PulJM), and neopullulanase (NeoJM) were selected for further studies. The second, third, and fourth stages of this study involved the protein expression, purification, and biochemical characterization of recombinant AmyJM, PulJM, and NeoJM, respectively. Purified AmyJM, PulJM, and NeoJM were optimally active at pH 6.5 and 40 °C. Interestingly, all three enzymes were found to degrade both raw and gelatinized forms of starches, producing a mixture of reducing sugars (i.e., maltose and maltotriose). Regarding raw starch hydrolysis, all studied enzymes demonstrated their highest efficiencies (35–51 % degradation) in hydrolysing raw wheat starch. The fifth stage of this study presented the optimisation of reducing sugars production from raw wheat starch using a combination of AmyJM, PulJM, and NeoJM through a 23 full factorial design and a central composite rotatable design. The optimised enzymatic cocktail's (36 U AmyJM, 41 U PulJM, and 45 U NeoJM) successfully improved the raw wheat starch hydrolysis and reducing sugars production by 57 % and six-fold, respectively, compared to the unoptimised condition. In conclusion, the findings suggest that AmyJM, PulJM, and NeoJM could be promising biocatalysts in direct raw starch saccharification processes.

Text 122660.pdf Download (1MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18628

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