Structure And Function Of The Novel Xylanases And Ca²⁺-dependent Carbohydrate Binding Modules

With the decrease of the available fossil resources and the increasing demand for energy,the development of new bioenergy to replace the fossil energy is especially imperative.Xylan is an important component of hemicellulose with the content next to cellulose,which accounting for 20%to 30%.According to the structure,xylan can be classified into?-1,4-xylan and?-1,3-xylan.Xylanase plays a key role in the degradation of xylan.Therefore,the researches on xylanase will help for the potentially use of renewable biological resources,which is expected to alleviate the problem of energy shortage.Based on this,the main works are listed below.Firstly,a novel carbohydrate binding module?CBM?was identified in a?-1,3-xylanase?Xyl3088?from Flammeovirga pacifica,which showed only 25.0%sequence identity with the reported CBMs with the coverage of 36.4%.To verify its function,a truncated?-1,3-xylanase?Xy13088-T?and a carbohydrate binding module?CBM3088?were expressed and purified.The thermostability and catalytic efficiency of the Xy13088-T declined significantly when compared with the full-length one,with the decreasing of the half-life and catalytic efficiency?Kcat/Km?by 90%.Interestingly,the CBM3088 showed the binding ability to?-1,3-xylan only when Ca²⁺existed,which was different from the reported CBMs of?-1,3-xylanases.The maximum amount of CBM3088 binding to?-1,3-xylan was 9.65?mol/g of?-1,3-xylan.The residues probably involved in the binding to the?-1,3-xylan and Ca²⁺were addressed by bioinformatics analysis.This was the first report of a Ca²⁺-dependent CBM in?-1,3-xylanase.Secondly,as the role of?-1,3-xylanase in biofuels and the functional xylooligosaccharides from some algal cell walls became more and more important.However,the preparation of pure?-1,3-xylanase is difficult.Besides,the low reuse rate and difficult to separate the free enzymes from the products result in high cost.These problems can be effectively solved by immobilized enzyme technology.Based on the?-1,3-xylanase?Xyl3088?,which is the most efficient catalytic activity found in our laboratory,HCP_pitch-CH₂�IDA was synthesized as an immobilized carrier by using cheap asphalt as the raw material,which achieved the separation,purification,and immobilization of the target enzymes in one step.The immobilization yield was about?90.01�1.05?%and the activity recovery was?80.66�0.87?%.The half-life of the immobilized?-1,3-xylanases was 50.30 min at 50�C,which was significantly longer than the free ones with 13.79 min.In terms of potential application,the immobilized?-1,3-xylanase could retain 65%of the original activity after stored at 30?for 80 hours and 87%of the original activity after 15 cycles of reuse.When?-1,3-xylanases were applied for the manipulation of algae Caulerpa lentillifera,the immobilized enzymes could release 30%more reducing sugar in 85 h.Thus,they may have great potentials in the algae biomass manipulation.Thirdly,functional annotation of the deep-sea bacterium Flammeovirga pacifica strain WPAGA1 genome revealed a coding gene Xyl4513,which had around 60%sequence identity with the verified?-1,4-xylanase?Clostridium saccharobutylicum xynB,ID:P17137?.Plasmids were constructed by gene synthesis and then expressed in E.coli BL21?DE3?,the pure enzymes were isolated by nickel column for the following studies.The results showed that the?-1,4-xylanase Xyl4513 had two conserved domains consisting of a catalytic module belonging to the glycoside hydrolase family11?GH11?and a carbohydrate binding module belonging to the CBM family 60.This is a rare phenomenon that the family GH11 xylanase contains CBM.The optimum temperature and pH of the purified Xyl4513 were 30�C and 3.0,which indicated Xyl4513 belonged to the low temperature and acidic?-1,4-xylanase.The truncated?-1,4-xylanase?Xy14513-T?and carbohydrate binding module?CBM4513?expression vectors were also constructed,respectively.After purification,the optimal temperature and pH of Xyl4513-T were 20�C,4.0 and the catalytic efficiency?Kcat/Km?was reduced 20%.Besides Ca²⁺,Mg²⁺,Ni²⁺,and EDTA could promote the catalytic activities of them,and the effect of Ca²⁺was more obvious.When containing 20mmol/L Ca²⁺,CBM4513 had a specific binding ability to?-1,4-xylan,and the maximum binding amount was 9.13?mol/g,which proved CBM4513 was a Ca²⁺-dependent CBM.Finally,the?-1,3-xylan was hydrolyzed by?-1,3-xylanase,and the composition of?-1,3-xylooligosaccharide was analyzed by thin-layer chromatography?TLC?and high-performance liquid chromatography?HPLC?.The biological activity of the oligosaccharide was determined.The results showed that xylobiose and xylotriose were the main active components in the?-1,3-xylooligosaccharide produced by the enzymatic hydrolysis.Among the four in vitro antioxidant systems,the antioxidant capacity of?-1,3-xylooligosaccharide showed a good dose-effect relationship in 0.25-2 mg/ml.For example,in the hydroxyl radical system,the scavenging activity of 0.25mg/ml Vc and?-1,3-xylooligosaccharides were 68.01%and 78.03%,respectively.The clearance rates were 99.91%and 95.45%at 1.5 mg/mL,while the scavenging activity of?-1,4-xylooligosaccharide at 2 mg/mL was only 5.47%,which was significantly lower than that of the?-1,3-xylooligosaccharide.The inhibitory effect of?-1,3-xylooligosaccharide on human breast cancer MCF-7 cells was detected by CCK8.The results showed that the concentration was 300-1000?g/mL,the time was 24-72 h,the higher the concentration,the longer the incubation time,the better the inhibition of the MCF-7 cells activity.This study not only experimentally verified a novel?-1,4-xylanase and two novel Ca²⁺-dependent CBMs,but also explained its mechanism by means of bioinformatics tools.Besides,the stability and recyclability of the?-1,3-xylanase were improved by immobilization technology.Finally,the functional bio-active?-1,3-xylooligosaccharide was prepared by enzymatic hydrolysis,which provides an alternative of enzymes for biofuels and the utilization of seaweeds.