Isolation, Characterization Of Thermophilic Bacterium DG-3 And Its Alkaline Pectinase Gene Expression In E.coli

Pectinases comprise a group of enzymes that catalyze the breakdown of pectin which exist in plant fibers.Nowadays the acidophilic pectinases have extensive applications in the extraction and clarification of fruit juices and wine.Alkaline pectinases are those which can cleave a-1,4-galacturonosidic linkages of pectic substances at alkaline conditions.Alkaline pectinases have been used in the purification of plant virus and bleaching of paper pulp.They are also widely used for cotton textile processing,degumming of plant bast fibers,treatment of pectic wastewaters.The application of alkaline pectinases will improve the traditional process of cotton textile pretreatment,which not only decreases the environment pollution but also reduces the consumption of industry water.In this paper,a bacterial strain producing alkaline pectinase was obtained from the high-temperature compositing sample.It was a Gram positive rod bacterium with round and hoar colony on plate.16SrDNA sequence of the strain showed 100%identity with that of Bacillus licheniformis.This bacterium was primarily identified as a strain belonging to Bacillus,named as DG-3.The temperature range for its growth was 15℃～55℃,with optimum 50℃.The alkaline pectinase activity produced by the DG-3 was 14.87U/mL, suitable temperature ranged from 60℃～70℃,and the suitable pH ranged from 9.8～10.5. The optimum temperature and pH of PGL were 65℃,10.0 respectively.The pectate lyase gene pel A from Bacillus licheniformis DG-3 was cloned and sequenced.The gene pel A from DG-3 was 100%similarity with that from B.licheniformis 14A.The pel A was expressed in E.coli as fusion protein.It was showed that the recombinant alkaline pectinase activity was 12 U/mL.Enzyme activity was promoted by 4～8 mmol/L Ca²⁺ ions.The genomic DNA of the strain DG-3 was extracted.The promoter library was constructed in E.coli DH5a with Puc-mpd as the promoter probe vector by shotgun-cloning method.36 positive clones,q1～q36 were obtained.Their methyl parathion hydmlase activity was assayed to compare the strength of the promoters.The results showed that the promoter of q26 was the strongest with MPH activity of 1259mU/mL.The promoters from q1,q8 and q26 were sequenced and subjected to online promoter prediction.