Study On Saccharification Of Brwon Algae By Alginate Lyase

As the third generation of biomass energy,brown algae biomass energy has been received more and more attention in the world at the background of fossil energy shortage and global warming.Compared with terrestrial biomass energy production,the production of brown algae biomass neither occupies the arable land,nor appear a competitive situation between the energy development and the food supply.In addition,brown algae biomass has many advantages,including high photo synthetic conversion efficiency,high carbohydrate content,lignin free,and easy to be saccharified.Alginate is the major carbohydrate of brown algae,its ethanol conversion efficiency is one of the main factors to affect the conversion efficiency from brown algae to bio-ethanol at the present stage,and the pretreatment of alginate saccharification impacts the conversion efficiency of the ethanol directly.In this paper,enzymatic pretreatment of alginate as the breakthrough point,the saccharification pretreatment of alginate were studied.The two-step saccharification pretreated technology of Sargasso used dilute sulfuric acid and alginate lyase was studied,and the condition optimization of the pretreatment was carried out.The saccharification efficiency of Sargassum reached 40%(w/w)after optimization.The saccharified Sargassum is fermented with industrial yesist,Saccharomyces cerevisiae R1-11,and the ethanol yield reached 91.9 g ethanol/kg substrate.A bacterium BP-1 which has the ability to secreting alginate lyase was screened from rotting Sargasso for the hydrolysis of the alginate.It was identified as S.haliotis BP-1 after Physiological,biochemical analysis combined with 16S rDNA analysis.Subsequently,the fermentation condition of strain BP-1 were optimized.The maximum yield of the alginate lyase was 30.4U/ml after optimization.All analysis showed that the strain BP-1 could use sodium alginate,agarose,soluble starch,and laminarin as carbon source.In addition,the strain BP-1 had the capability of extracellular electron transfer,could transfer the electrons from the cell surface to Mn(Ⅳ),and reduced Mn(Ⅳ)(brown)to Mn(Ⅱ)(colorless).It might be as a potential bacterium to manufacture microbial fuel cell and resume heavy metal pollution.The alginate lyase(crude enzyme)produced by strain BP-1 was used to saccharify sodium alginate and kelp,which showed that sodium alginate could be hydrolyzed into unsaturated single uronic acid by the crude enzyme,and the crude enzyme could enhance the saccharification efficiency of kelp mash significantly.It not only could reduce the viscosity of the kelp mash greatly,but also increased the efficiency of the dissolution of mannitol from kelp cells.The dry weight of kelp was decreased 70.96%(w/w)after pretreated with crude alginate lyase,54.17%(w/w)higher than the control;the extraction yield of mannitol was 17.23%(w/w);the reduce sugar yield was 14.81%(w/w).The kelp which pretreated with crude alginate lyase was used to ferment with P.angophorae,the ethanol yield was 7.83%(v/w).The result of ethanol fermentation used kelp saccharification liquid and sodium alginate saccharification liquid shown that a majority of microorganisms which are able to produce ethanol can not use the unsaturated monosaccharide of alginate degradation products.The genomic DNA of strain BP-1 was sequenced,and the genetic framework map was constructed.The analysis shown that the genome of strain BP-1 is a simple genome,there is no contamination with other species.The size of secquenced genome is 3.64M,the concentration of GC is 53.91%.The result of gene annotation shown that the genome has 3222 encoding gene,including the encoding gene of key enzyme involved in hexose diphosphate pathway,citric acid cycle,Pentose phosphate pathway and Entner-Doudoroff pathway.In addition,it has the encoding gene of key enzyme of alginate metabolism.Furthermore,it has the encoding gene of alginate lyase,agarase,cellulose,amylase and some protein involved in extracellular electron transfer.The ability of extracellular electron transfer and polysaccharide metabolize further need to be validated.It is interesting that strain BP-1 have an encoding gene of alcohol dehydrogenase,indicates that strain BP-1 have the ability to producing ethanol under certain conditions.Purification and identification of the alginate lyase secreted by strain BP-1 was performed.The result shown that the alginate lyase from strain BP-1 was a multifunctional enzyme,it had the activity for poly(M)and poly(G)enzymolysis;Its the molecular weight is about 70kDa;the optimum reaction temperature is 45℃,it is stable at 25-35℃;The optimum reaction pH is 7.5-8.0;The enzyme activity could be inhibited by a majority of metal ion,SDS and EDTA.A certain concentration of NaCl could enhance the activity of the enzyme.The N-terminal sequences of alginate lyase and genome sequences of strain BP-1 was analysised,the results showned that the encoding gene of alginate lyase is 2157bp,the encoding proteins is 718aa,the size of signal peptide is 26aa。...