Structural Analysis Of The Mutant Of Thermostable Endogiucanase And The Functional Evolution

Enzymatic conversion of the polymer to individual sugar units was a main focusof the biotechnology industry. Discovering the effective and stable cellulolyticenzymes, and analyzing the relationship of structure, function and evolution havebeen the research focus of enzymology and biofuel. The thermostable cellulases fromCaldicellulosiruptor bescii had been systematically studied in our lab. One of themost importmant number CbCel9A belonged to GH9family is a multi-domain,processive endocellulase, which has some outstanding characteristics, such as, heatsteadiness and high activity on microcrystalline cellulose. Basing on the analysis ofhomology of sequence and structure, a non-conservative loop constructed by18aminos is located in the end of the avtive cleft. The flexibility and compostion of thisloop may have an effect on enzyme catalytic functions, so a series of truncatedmutants was engineered to clarify the roles of the loop in CbCel9A. The catalyticefficiency, stability mechanism and intramolecular synergy of CbCel9A were statedby structural Analysis of the mutant, and then constructing and analysing thefunctional sites in active site architecture of GH9family glycoside hydrolase bymeans of bioinformatics, which might be useful for cellulase engineering. So themainly research work in this thesis are as follows:(1) Functional characterization of the loop mutants. Firistly, a series of truncatedmutants was engineered based on the length of the loop, named as N2, N4, N6, N8toN18. The most significant enzymology change was observed in mutant N8, such as,the activity on Avciel of N8was1.91times higer than that of the wild type, the valueof Kmdecreased to61%of the wild type enzyme when hydrolyzing RAC in kineticsexperiments, the processivtiy on filter paper increased from5.81to11.28, and the halflife time increased from12h to16h at85℃.(2) Structural Analysis of mutant N8. Under the crystallization condition of0.2MCalaium acetate hydrate pH6.5,0.1M Sodium cacodylate trihydrate,10%W/V Polyethylene glycol8,000, the three-dimensional structure of the mutant N8with2.1was determined. The endoglucanase, N8, is comprised of a family9catalyticdomain attached to a family IIIc cellulose-binding domain by a tight linker with only18amino acids. The two domains together form a flat platform onto which crystallinecellulose is suggested to bind and be fed into the active-site cleft for endolytichydrolysis. Comparing to PDB:4DOE protein which only possessed CD domain, thenon-reducing end of the active center cleft in mutant N8becamed relatively open,extending the substrate binding space. The more electronegativity area was exposed,making for the enzyme-substrate binding, which is consistent with the low Kmvalueof the mutant N8on RAC. And the postion of catalytic acid site E420was changedand the distance between E420and catalytic base site D58increased from5.49to6.80, which may increase the hydrolysis effiency.(3) The stability mechanism of CbCel9A and mutant N8. The optimum reactiontemperature of CbCel9A and mutant N8are all above80℃as contrast to the enzymecontaing CD domain with the optimum reaction temperature is only50℃, indicatingthat CBM domain has important effect to the stability of the whole structure.Hydrogen bonding, hydrophobic interaction are important driving force for CD-CBMinteraction to a rigid construction. The DSC experiment for the mutant N8with asingle peak also shows the integrity of complete structure, which the two domainsforms an entirety structure.(4) Active-site architecture and function evolution relationship analysis in GH9family. According to the co-crytal structure of enzyme and substrate (PDB:4TF4)form Thermobifida fusca, we found out the potential amino acid sites consisted in theactive site architecture of GH9family glycoside hydrolase. Based on the homologymodelling and structure alignment, we calculated the sequence conservation of theamino acid sites and the frequency of occurrence of the amino acid tape in the cleftcompared to the whole family. Tryptophan, tyrosine, aspartic acid, histidine andasparagine were with very high occurring frequency. There were seven conservedsites in the active-site cleft, besdides W260located in the-2subsite, all the concervedsites around the-1to+1subsites, indicating that the three dimensional frameworkconstructed by the concerved sites may be the fundamental structure foundation forhydrolyzing the glycosidic bond in GH9family glycoside hydrolase. We thought thefunction of the conserved site D55near to D58was not only assisted to catalyze but also to maintain the position and conformation of sugar ring in-1subsite, and thesecond function mighe be the most important. The no-conserved site H120contracting with D58by one hydrogen bond might impact the optimum reachtion pH.In this papar, we the systemically and widely studied the function and structure ofCbCel9A and mutants, clarifying the organizational characteristics of the CD andCBM domain in CbCel9A, revealing the important influence of catalytic center forenzyme catalysis, thermostability, and substrate binding ect.. Constructing theActive-site architecture in GH9family, which is important for clarifying the functionand action mechanisms of cellulase and useful for protein engineering.