Identification of Locally Isolated Cellulolytic Stenotrophomonas maltophilia from Rice Straw and Optimization of its Cellulase Activity

A highly cellulolytic bacterium was locally isolated from rice straw and identified as Stenotrophomonas maltophilia. Identification of the isolate based on the morphological, cultural and biochemical characteristics was confirmed with 16S rDNA analysis. The bacterium showed the highest level of reducing sugar and extracellular protein production when incubated for 3 days (348.75 μg/ml and 288.5 μg/ml respectively) at 40°C temperature (463.0 μg/ ml and 333.0 μg/ml respectively) and pH 6.5 (360.0 μg/ml and 349.0 μg/ml respectively) in Winstead’s broth having 1.5% CMC and 0.2% Yeast Extract as carbon and nitrogen source respectively. Crude cellulase enzymes produced by the bacterium showed the highest CMCase activity rather than FPase, Avicelase and ²-Glucosidase activities. Cellulase activity of the crude enzyme was also determined using the same parameters. The crude cellulase enzyme showed the highest CMCase activity when incubated for 60 minutes (232.5 U/ml), at pH 6.5 (105.0 U/ml), 35°C temperature (69.75 U/ml) using CMC and Peptone as carbon and nitrogen source respectively. Crude cellulase showed the highest activity in presence of mercury and SDS as metal and detergent respectively. Substrate specificity and SDS-PAGE analysis reveals that the cellulase may be an endo-1,4-glucanase. Bangladesh J Microbiol, Volume 37 Number 1 June 2020, pp 15-22

Efficient expression vectors and host strain for the production of recombinant proteins by Yarrowia lipolytica in process conditions

Background The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters. Results The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL−1) within 24 h as compared to the mono-copy strain. Conclusions The expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica.