Characterization,Thermostability Engineering and Application of the Chitinases from Paenibacillus Pasadenensis

Author:Xu Pei

Supervisor:lou wen yong


Degree Year:2019





Chitin is the most abundant organic biopolymers second to cellulose in nature.N-acetylglucosamine(GlcNAc),the hydrolysate of chitin,has been proved to play a significant role in the field of functional food and biophamarcy.In general,chitin and its derivative are produced in a traditional way using concentrated acid,probably leading to environmental pollution.It is very significant to explore a biocatalytic method.Chitinase has been proved to specially catalyze the hydrolysis of chitin to chito-oligosaccharide and GlcNAc under moderate condition,however,chitinase shows poor thermostability under high reaction temperature.In addition,the compact structure of chitin hinders the accession of chitinase to chitin substrate,causing low catalytic efficiency.Therefore,from a perspective of application,it is of great environmental and economical value to develop an efficient approach to prepare GlcNAc based on chitinase biocatalysis to replace the traditional chemical way.From a standpoint of science,the two key issues to be addressed in this work are:1)to figure out the relationship between the structure and function of chitinase;2)to further improve the enzymatic process efficiency by solvent engineering regulation.The main results of this dissertation are listed as following:1.To solve the source of chitinase,a strain which could efficiently degrade chitin was isolated.By morphological,physiological,biochemical and biological test,the strain CS0611 was identified to be Paenibacillus pasadenensis and named Paenibacillus pasadenensis CS0611.After growing in an optimized culture medium for two days,the strain could use up the crab shell particles,which was served as the only carbon source.The resultant chitinase achieved an activity of 798.6±16.6 U/L。2.To settle the problem of long incubation period and tedious purification of enzyme in a fermentation process using natural microorganism,the chitinase genes were cloned and heterogenously expressed in E.coli.By measuring and analyzing the genome data of Paenibacillus pasadenensis CS0611,four chitinases with a molecular weight of 65.4 kDa,45.7 kDa,38.1 kDa and 42.4 kDa,respectively,were identified.The four chitinases were then ligated to pET-28a to construct recombinant plasmids.PpChi1,PpChi2 and PpChi4were expressed in E.coli BL21(DE3)with good solubility and purified with a specific activity of 3.34 U/mg,2.96 U/mg and 2.22 U/mg,respectively.PpChi3 was expressed as inclusion body even though the expression condition was optimized and its specific activity was 3.14 U/mg.The optimum reaction temperature of PpChi1,PpChi2 and PpChi4 was found to be 45 oC,55 oC and 50 oC,respectively.The three chitinases had a common optimum reaction pH of 5.0,and showed excellent pH stability at a range of 4.0-10.0.Various metal ions exhibited different effects on the three recombinant chitinases.SDS could cause an absolute loss of activity of the tested chitinases.EDTA,Tween-80 and TritonX-100 didn’t show any inhibitory or stimulative effect,suggesting the chitinases were not metalloenzyme.β-Mercaptoethanol could lead to the activity loss of PpChi1,but had no effect on PpChi2 and PpChi4,suggesting thatβ-mercaptoethanol might destroy the disulfide bond(41C-61C,305C-316C)in PpChi1.All the three chitinases showed excellent affinity to chitin powder and colloid chitin.The TLC test of the product of colloid chitin catalyzed by the chitinases displayed that(GlcNAc)2 was the main product of PpChi1 and PpChi2 and GlcNAc was the only final product of PpChi4.PpChi1/PpChi2 and PpChi4could synergetically hydrolyze colloid chitin to GlcNAc.3.As for the poor thermostability of PpChi1,a semi-rational design was used to engineer the thermostability of PpChi1 based on its amino acid sequence and 3D structure.By single site mutation and combinatorial mutation,mutant PpChi1-S244C-I319C/T259P(M3)showed a significant improvement of 26.3-fold(5.5 min vs 150 min)of half-life at 50 oC compared to wild-type PpChi1.The specific activity also increased by 1.3-fold.The optimal reaction temperature of M3 had an increase of 7.5 oC and the optimal reaction pH kept 5.0.The kinetic parameters Km,kcatat and Vmax were 1.68mg·mL-1,941.2 min-1,and 13.4μmol·min-1·mg-1,respectively.The catalytic efficiency improved by 49.5%.By structural and molecular dynamic simulation analysis,the good thermostability of M3 was attributed to its improved structure rigidity at high temperature.4.Regarding the low hydrolysis efficiency of chitin in an enzymatic process,caused by high crystalline index and poor solubility of natural chitin,a pretreatment step by five kinds of ionic liquids and three deep eutectic solvents(DESs)was investigated.The[BMIm]Ac-pretreated chitin showed the best hydrolysis efficiency,while DESs didn’t show any effect.The results of SEM,FTIR and XRD suggested that chitin pretreated by[BMIm]Ac and[EMIm]Ac had significant structural change(e.g.surface morphology)after the pretreatment and regeneration process.The hydrogen bonding network changed very much,leading to a decrease of crystalline index of chitin,which could benefit for the access of chitinase to the molecular chain of chitin and a high hydrolysis efficiency.A strategy of combining two chitinases in the hydrolysis of[BMIm]Ac-pretreated chitin was performed.The combinations of M3+PpChi4 and PpChi2+PpChi4 afforded a yield of GlcNAc by786.6±16.5 mg/g chitin and 640.8±15.1 mg/g chitin,respectively.By altering the sequence of adding different chitinases into the reaction system,the yield of GlcNAc increased up to864.5±14.5 mg/g chitin.Additionally,the hydrolysis of pretreated chitin could afford a yield of GlcNAc for 614 mg/g chitin by using the recovered[BMIm]Ac for 8 times.Therefore,chitin could be efficiently transformed to GlcNAc through a pretreatment process using ionic liquid.