Metabolomics approach used for understanding temperature-related pectinase activity in Bacillus licheniformis DY2.

Pectinases are enzymes that catalyze pectin degradation. There is a global demand for pectinases because of their wide utility and catalytic efficiency. Optimization of the fermentation process to increase the pectolytic enzyme activity is generally practiced to lower process costs, but whether temperature influences the metabolome, enhancing pectinase activity, is not known. Here, we developed a metabolomics approach to explore it. The activity of P-DY2 pectinase produced by Bacillus licheniformis DY2 was higher in cells grown at 30°C than those grown at 37°C. Differential metabolome analysis revealed fluctuating tricarboxylic acid (TCA) cycle at 30°C. Consistently, the transcripts of TCA cycle genes and activities of pyruvate dehydrogenase and α-Ketoglutaric dehydrogenase were lower at 30°C than 37°C. Furthermore, inhibition of pyruvate dehydrogenase and succinate dehydrogenase enhanced the activity of P-DY2, supporting the conclusion that the inactivated pyruvate metabolism and TCA cycle were required for pectinase activity, and that P-DY2 was TCA cycle-independent. Collectively, these findings indicated that fermentation temperature affected P-DY2 activity by metabolic modulation, with an inactivated TCA cycle as a characteristic feature of high P-DY2 activity. More importantly, the present study highlights an approach of promoting pectinase activity through metabolic modulation by using metabolic pathway inhibitors.

Ultrasound assisted process intensification of uricase and alkaline protease enzyme co-production in Bacillus licheniformis.

Low energy ultrasound irradiation was used to enhance co-production of enzymes uricase and alkaline protease using Bacillus licheniformis NRRL 14209. Production of uricase and alkaline protease was evaluated for different ultrasound parameters such as ultrasound power, time of irradiation, duty cycle and growth stage of organisms at which irradiation is carried out. Maximum uricase production of 0.825 U/mL and alkaline protease of 0.646 U/mL have been obtained when fermentation broth was irradiated at 6 h of growth stage with 60 W power for 15 min of duration having 40% of duty cycle. The enzyme yield was found to be enhanced by a factor of 1.9-3.8 and 1.2-2.2 for uricase and alkaline protease respectively. Nevertheless, intracellular uricase was also observed in a fermentation broth after ultrasonic process intensification. The results indicate the effectiveness of low frequency ultrasound in improving enzyme yields with a vision of commercial applicability of the process.

A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.