Isolation and molecular identification of cellulolytic bacteria from Dig Rostam hot spring and study of their cellulase activity

Cellulose is the main structural component of lignocellulosic wastes that can be converted to sugars and biofuels by cellulase.Due to wide applications of this enzyme in various industries around the world,cellulase is considered as the third industrial enzyme.The ability of thermophilic bacteria in the production of heat-stable cellulases has made them valuable tools in biotechnology.The aim of this study was isolation and molecular identification of cellulolytic thermophile bacteria from Dig Rostam hot spring and investigating their cellulase activity.Samples were taken from water and sediments of this hot spring,and cellulolytic bacteria were enriched in media containing cellulose as the only carbon source.The bacteria were incubated at 60℃,and single colonies were then isolated on solid media.Congo red assay was used as a quick test for the qualitative screening of cellulase activity.According to these qualitative results,four colonies named CDB1,CDB2,CDB3,and CDB4 were isolated,and their growth curve and some other characteristics were determined by biochemical assays.Moreover,endoglucanase,exoglucanase,and FPase activities of the isolates were investigated quantitatively.Results indicated that CDB1 exhibited the highest endoglucanase(0.096 U/mL)and exoglucanase(0.156 U/mL)activities among other isolates.16S rDNA partial sequencing indicated that CDB1 had 99%similarity to the genus Anoxybacillus,and the other isolates showed the highest similarity to the genus Geobacillus.The cellulase gene of CDB1 isolate with the highest cellulase activity was also cloned,and its sequence is reported for the first time.Further studies on this thermophilic enzyme might be useful for industrial applications.

A Method for Determination of Hexokinase Activity by RP-HPLC

A method was established to determine the activity of hexokinase by reverse-phase high-performance liquid chromatography （RP-HPLC）. The activity of hexokinase was determined by detecting the amount of ADP in the reaction that converts glucose into glucose-6-phosphate. The separation degree of ATP and ADP was as good as 3.46 （separation degree^l.5）. In the range of 0.01-0.40 mmol/L, there was a good linear relationship between concentration of ADP and its peak areas （the correlation coefficient was 0.999）. The relative standard deviation（RSD） of intraday was 1.43%-1.46%, that of interday was 2.12%-2.15%. At 30℃ （optimal temperature） and pH7.0 （optimal pH）, the recovery rate of hexokinase was 95.3%-97.6%. The results showed that the method had good precision and high recovery rate, and the enzyme activity was determined through only one-step reaction. To some extent, this method can reduce the cost.