Study On The Functional Role Of The Gene Chu₁279 Located Within The Putative PUL Cluster In Cellulose Degradation By Cytophaga Hutchinsonii

Lignocelluose is the most abundant renewable resources on the earth.Its high-efficiency conversion to fermentable sugars that can be further transformed to biofuels and bio-based chemicals holds great potential for sustainable development of human society.Various microorganisms in nature can degrade cellulose.Among others,members of Bacteroides encode the start utilization system?Sus?system and the Sus-like system by polysaccharide utilization locus?PULs?gene cluster to realize the degradation,absorption and utilization of polysaccharide substrates.Cytophaga hutchinsonii?Chutchinsonii?is an aerobic gram-negative bacteria,belonging to Bacteroides,which can efficiently degrade insoluble crystalline cellulose in a cell contact-dependent manner.C.hutchinsonii does not secrete extracellular free cellulases nor does it form cell associated cellulosome.C.hutchinsonii lacks obvious carbohydrate binding modle in cellulases.It remains unclear with respect to its degradation mechanism of crystalline cellulose.It is speculated that C.hutchinsonii may achieve efficient cellulose utilization on the basis of a third new cell-bound cellulose-degrading enzyme system.But,until recently,little was known about its mechanism of cellulose utilization.It has been reported in our laboratory that C.hutchinsonii may have a polysaccharide like utilization locus,CHU₁276-CHU₁280,similar to the known PUL in Bacteroides,i.e..Outer membrane proteins,CHU₁276 and CHU₁277 are encoded by genes that play an important role in cellulose degradation.CHU₁280 is presented suggesting that it may be a processive endoglucanase.It is speculated that uncharacterized protein CHU₁278 and CHU₁279 may function significantly in the process of cellulose degradation.In this regard,the paper was conducted focusing on the specific functions of CHU₁279 during crystalline cellulose degradation by C.hutchinsonii.The main research content is described as follows:1.CHU₁279 is key to C.hutchinsonii deploymerizing and assimilating crystalline cellulose in study of ?1279 characterization.Gene deleted mutant strain ?1279 which was constructed in this study was constructed to measure the growth,cellulase activity and cellulose adsorption properties of A1279 and wild-type?WT?under different carbon sources,so as to analyze the phenotypic differences of the mutant.In the growth analysis,the mutant cannot grow in medium with Avicel as the sole carbon source,A1279 loss of the utilization ability of crystalline cellulose.The absence of CHU₁279 also reduced the cellulase activity by 25 percent and adsorption efficiency of crystalline cellulose by 30 percent.However,the CMCase activity and pNPG activity in the cytoplasm were basically unchanged compared with the wild type.2.Expressing and purifying the recombinant CHU₁279 protein and the study shows that CHU₁279 can adsorb on crystalline cellulose.In this study,the full-length recombinant protein CHU₁279 were constructed and purified in E.coli by affinity chromatography,to detect the role of the recombinant protein in water insoluble polysaccharides adsorption.According to the results,CHU₁279 was such a protein that can specifically adsorb crystalline cellulose and weakly bound with xylan.Analysis shew that CHU 1279 may consist of two domains.We broke CHU₁279 into independent parts,which C-terminal domain CHU₁279-2 had the adsorption function of crystalline cellulose,with unclear function of N-terminal CHU₁279-1.3.Constructing the point mutants,Indicating that Tyr253?Phe256 and Phe343 are the key amino acid sites of CHU₁279 in crystalline cellulose binding.To investigate the roles of Trp89?Phe92?Phe180?Asn182?Tyr253?Phe256?Phe343?Asn345 in ligand binding,each residue was individually mutated to alanine.The mutants were expressed and purified in E.coli.Exploring the difference of crystalline cellulose adsorption efficiency between mutant protein and WT.The results indicated that Y253A,f256 and f343 lost the ability to bind crystalline cellulose.The ability of binding crystalline cellulose was slightly reduced in Mutant N345A.There is no change in binding process of mutant W89A?F92A?f1 80 and N182A.In addition,there was no change in secondary structure of protein between mutants and WT in CD spectra.In summary,Tyr253,Phe256,Phe343 and Asn345 were key amino acid sites of CHU 1279 absorbing crystalline cellulose.