| Cellulose, the structural component of plant cell walls, is a linear polymer of glucose molecules, linked to one another primarily by β-1,4-glycosidic bond. It’s the most abundant renewable resources which can be converted to glucose by β-glucosidase. Cellulosic biomass is the most potential renewable resource. In cellulose degradation, the breeding of high-efficient cellulases and cellulase-producing strains have become the focus of research globally.A cellulose degrading microorganism, named as Thermobifida alba F-7, was isolated from high temperature compost and simply identified by its16S rDNA in our lab. It could produce cellulase and xylanase with characteristics of high temperature tolerance. Due to few relevant reports to understand this strain deeply we first studied its colony morphology systematically. Thermobifida alba F-7grows in clusters with white aerial mycelium dichotomously branched with nonfragmenting hyphae. Its single spores, oval to round, are borne on dichotomously branched sporophores, resulting in spore clusters on aerial mycelium. It is Gram-positive and can produce many kinds of extracellular enzymes, such as protease, rennet, cellulase, amylase, and xylanase. All of these characters are in accordance with Thermobifida alba. The characteristics of the crude enzymes of Thermobifida alba F-7were studied. The optimal pH of the cellulase and xylanase were neutral lean to acidic, however they retained their activity after being processed in buffer between pH4to pH10for one hour, indicating the good acidic and alkali resistance of the crude enzymes. The optimal temperatures of cellulase and xylanase are about60℃and70℃, respectively. No much change had been detected after being incubated in60℃for one hour, indicating a good thermal stability of the crude enzymes as well. The characteristics of the crude enzyme show a potential industrial application.The fermentation conditions were optimized by One-factor-at-a-time design, Factorial design, Steepest ascent design and Response surface methodology. First, the best carbon source,1%composite carbon source (wheat bran:corncob residue=1:1), and the best nitrogen source were screened by One-factor-at-a-time design, resulting the yield of cellulase and xylanase improved8and10times respectively. Then, three key factors of carbon source, corn syrup powder and Na2HPO4were screened out by Plackett-Burman design, and a reliable fermentation model of T. alba F-7was finally set up by RSM. According to the predicted optimal combination for cellulase fermentation, the yield of cellulase increased to13.4IU/mL compared with the original1.2IU/mL, which was about7.5times improvement.Thermobifida alba F-7was also mutated by traditional ultraviolet mutagenesis method to increase its yield of enzymes. Four mutants (UV-59, UV-114, UV-134and UV-148) with genetic stability were obtained. Compared with the wild type strain, the mutants showed about50%higher yield of enzymes.The crude enzymes of Thermobifida alba F-7and its four mutants were applied to the saccharification of corncob residue. The results showed that the main component of enzymatic hydrolysis liquid was cellobiose and relatively lower content of glucose, indicating that the content of cellobiase might be low in the crude enzymes. So, the improvement of cellobiases should be considered in future breeding. The four mutants showed better converting efficiencies of cellulose to reducing sugar compared with the wild type, about54%, which was still too low for industrial application. However, the concentration of glucose and cellobiose generated in the saccharification at50℃,55℃and60℃were similar, which indicated a good thermal stability of the crude enzymes and might be considered for special applications in the future. |
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