Screening, Constructing, Identification And Enzymatic Characteristics Of Cellulose-degrading Flora From Shipworms

Highly active cellulose-degrading bacteria are screened and isolated from two samples:one is shipworm collected in Port Arthur Saltern,Dalian,China,and the other is garden soil collected in Dalian Jiaotong University.Studis are based on the shipworm bacteria, supplemented by the soil bacteria with better activities,in order to lay a foundation for further degrading natural cellulose.Strains are cultured by CMC-Na or filter paper as sole carbon.Though experimental methods such as Congo red staining and disintegration of filter paper,six aerobic bacteria are screend from the homogenate of shipworm and garden soil.By rescreening of shaking flasks for12days,T14912from soil has the highest Cx activity-18.19U/mL,and Cx activities of S4713and S2321from shipworm are13.95,11.65U/mL, respectively.When the fermentations of shaking flasks reach the6th day,by quantitative measuring, shipworm bacteria S4713has four cellulase with different activities,and that is endoglucanase (Cx)>β-glucosidase (β-Gase)>holoenzyme (FPA)>exo-glucanase (C1).It shows that the holoenzyme activity is not simple summation of the other three enzyme activities,but the results of cellulose synergistic degradation for other three enzyme.The influences of pH and temperature on four cellulase activities of S4713show single peak curves,and the optimum pH and temperature are different.In general,the ranges of optimum pH and temperature are6.5～7.5,50～60℃for four enzymes,respectively.By pairwise constructing compound flora from six highly efficient cellulose-degrading bacteria,S2321and T14912have the most significant effects of collaborative enzyme production,and their Cx and FPA average activities are20.89,6.42U/mL,respectively.By flask fermentation for12days,the best time of Cx production is about4-7d,while the best time of FPA production is about4-8d for different fermentation broths.By morphological observation,physiological and biochemical characteristics,and phylogenetic analysis of16S rDNA,we learn that six bacteria are gram-positive with fimbriae.Their spores are oval,centered and not inflate;S3212and S4941are preliminarily identified as Bacillus Jlexus,while S4933is one of Bacillus flexus or Bacillus pumilus;Cormpared with the reference strain Bacillus flexus DSM1320T,S2321and T14912can be identified as Bacillus flexus,S4713is maybe identified as a new subspecies of Bacillus flexus.