Polyphasic Taxonomy Analysis Of Four Novel Marine Bacterial Strains And Studies On Agar-degrading Eneymes Produced By Strains HQM9~T And Q1~T

Energy,resources and environmental issues are the main bottlenecks to constrain socio-economic sustainable development in the 21st century.In order to solve the problem of energy shortage,many researchers turn to seek new bioenergy pathways from the ocean.Ocean area,which is rich in natural biological resources,accounts for more than 70%of the total area of the earth and contains a large number of algae which can be used to biomass conversion.Agar,as one marine functional polysaccharide,is the main cell wall component of marine red algae,so agar has huge amount of biomass and is one of the most important sources of biomass conversion.Besides,the agar-oligosaccharides from agar hydrolysis are water-soluble and easy to be absorbed by the body,so they have great prospects for the application in food,medicine,cosmetics and other fields.Microorganisms,as primary consumers of degradable agar,are the main species source to study the agar biomass conversion and preparation of agar oligosaccharides.Therefore,this paper made use of the bacterial agar degradation as the main line,and carried out a series of research works.First of all,four marine novel bacteria including two agar-degrading bacteria have been classified and identified in this study.One of them is a novel agar-degrading bacterium,designated QM50~T,isolated from coastal seawater in an aquaculture site near Qingdao,China.Strain QM50~T is Gram-stain-negative and has the ability to hydrolyse agar,gelatin,casein,DNA and Tween 80.The major respiratory quinone is Q-8 and major polar lipids are PG and PE.On the basis of phylogenetic,genome and phenotypic data,strain QM50~T represents a novel species of genus Colwellia,for which the name Colwellia agarivorans sp.nov.is proposed.Another strain is the other agar-degrading bacterium,designated HZ1~T,isolated from the surface of marine alga Porphyra yezoensis Ueda.Strain HZ1~T is Gram-stain-negative and has the ability to hydrolyse agar,gelatin,casein and Tween 80.The major respiratory quinone is MK-6 and major polar lipids are PE and three unidentified polar lipids.Based on the phylogenetic analysis and phenotypic properties,it is concluded that strain HZ1~T represents a novel species in the genus Tenacibaculum,for which the name Tenacibaculum agarivorans sp.nov,is proposed.The third strain is a moderately halophilic bacterium,designated SS9T,isolated from the gill homogenate of a shark.Strain SS9T is Gram-stain-negative and has the ability to accumulate PHA and grows optimally in the presence of 6.0%(w/v)NaCl.The major respiratory quinone is Q-9 and major polar lipids are PE and PG.Base on phylogenetic,MLSA,phenotypic and chemotaxonomic results,strain SS9T represents a novel species in a new genus in the family Halomonadaceae,for which the name Pistricoccus aurantiacus gen.nov.,sp.nov.is proposed.The forth strain is N211~T,isolated from marine sediment obtained off the coastal area of Weihai,China.Strain N211~T is Gram-stain-negative and has the ability to hydrolyse gelatin and Tween 80.The major respiratory quinone is Q-8 and major polar lipids are PE,PG and one unidentified polar lipid.It is evident from phenotypic data and phylogenetic inference that strain N211~T represents a novel species of the genus Thalssotalea,for which the name Thalassotalea sediminis.nov.is proposed.In addition,this study also completed the draft genome sequences of two agar-degrading bacteria.The full genome sequence of strain QM50~T is 4.7 MB and GC content is 35.8%.On the basis of annotations results of the Swiss-Prot database,strain QM50~T has five ?-agarase genes,four carragenase genes and one alginate lyase gene.The full genome sequence of strain HZ1~T is 5.6 MB and GC content is 30.9%.On the basis of annotations results of the Swiss-Prot database,strain HZ1~T has eleven ?-agarase genes,one a-agarase gene,two carrageenase genes and one alginate lyase gene.Besides,these two strains have been annotated different galactose metabolic pathways using KEGG database.Secondly,11 agarase genes and 6 NABH genes from two agar-degrading bacteria HQM9~T and Q1~T which were previously identified by our lab were studied in this study.Base on bioinformatics analysis,agarase AgaAJ belongs to the GH86 family,agarase Q1B2 belongs to the GH118 family and other nine agarases belong to GH16 family.All NABHs belong to the GH117 family.After exogenous expression of agar-degrading enzyme genes,we successfully constructed fifteen genetically engineered bacteria including eleven agarase genes and four NABH genes.Therein,nine agarase genes successfully expressed target proteins and exhibited certain agar-degrading activities,four NABH genes successfully expressed target proteins but failed to exhibit activity.By affinity chromatography and inclusion body renaturation,one recombinant agarase Q1B1 was successfully purified.After that,preliminary enzyme studies were conducted on the purified protein.The optimum substrate concentration,temperature and pH for the protein was agarose 1.6%,40? and pH 8,respectively.The protein has agar substrate specificity and the specific enzyme activity is 222.84 U/mg in the condition of agrrose 1.0%.In addition,DTT has a significantly positive effect on the activity.The degradation products of purified Q1B1 are neoagarotetraose and neoagarohexaose,and neoagarohexaose is the minimum degradation substrate.At last,this paper also studied the differential expression of agar degratation related genes on transcription level in bacterial different growth phase after agarose induction.The research results showed that concentrated expression of agar-degrading enzyme genes turned up in the early stage after strain was induced by agarose,and continued to decrease with growth of the strain.When the strain grew to late of stable phase,agar-degrading enzyme genes were basically stopped to express.In addition,according to the distribution of the agar-degrading genes in the genome,most of the agar-degrading enzyme genes in strain Q1~T may be individual regulation type.Although the genes expression is concentrated,the expression quantity is only related to the induction condition,but not with the position of the genes.