Secretory Expression In Bacillus Subtilis And Thermostability Of β-cyclodextrin Glycosyltransferase

According to the wild used of cyclodextrin in food, pharmaceutical and cosmeticindustries, the cyclodextrin glycosyltransferase (EC2.4.1.19, CGTase) used to productcyclodextrins industrially has become the focus of scientific research nowadays. In order toovercome the low productivity of CGTase with wild strains, the extracellular overexpressionof the enzyme in genetically engineered bacteria is one of the effective way. Currently, theEscherichia coli is normally used as the host bacteria during the extracellular expressing ofCGTase. However, during the E. coli expression, the CGTases were usually formedinsolubility inclusion bodies. Furthermore, CGTases generally have relatively lowthermostability, which severely limited their industrial application and the production ofcyclodextrins. Therefore, it is important to establish the thermostability of CGTases withpotential industrial roles. In this study, the gene encoding-CGTase from Bacillus circulansSTB01was expressed in B. subtilis, and the thermostability of the enzyme was evaluated, too.The main results are attached as follows:(1) The cgt gene encoding-CGTase from B. circulans STB01was cloned in a pSTvector. Then, the expression plasmid cgt/pST was constructed and transformed into the host B.subtilis WB600for extracellular expression-CGTase.The culture conditions was optimized in shaking flasks for extracellular fermentation ofthe recombinant-CGTase with B. subtilis hosted cgt/pST. The results showed that the seedsin mid logarithmic growth aged14h were best for fermentation. The initial medium wasterrific broth (TB) in pH7.0, after incubation at37oC for48h, the-cyclodextrin-formingactivity of the culture medium was27.9U/mL, which was nearly19-fold higher than that ofthe parent strain. Further optimized the carbon and nitrogen source of the TB medium,replacements the glycerinum in TB medium with6g/L corn starch and30g/L yeast extract asthe nitrogen source led the extracellular activity to31.2U/mL. The additions of leucine and/oraspartate improved the fermentation level obviously, and0.5mM Fe3+could furtherlyenhance the activity to36.9U/mL. To the best of our knowledge, this is the highest-cyclodextrin-forming activity of recombinant-CGTase expressed in B. subtilis.(2) The recombinant-CGTase could be purified by a combination of Phenyl HPhydrophobic chromatography and Q-HP anion exchange chromatography. The recovery ofthe enzyme was45.3%.The apparent molecular weights of the β-CGTase was about76.5kDa and presentedmonodisperse, which indicated that the purified enzyme was a monomer in solution. Theoptimum cyclization reaction pH of the β-CGTase was6.5, and showed more thermostable inglycine-NaOH buffer. The optimum cyclization reaction temperature was60oC. The half-lives (t1/2s) of the enzyme at45oC,50oC,55oC and60oC were about6.8h,4.5h,55.6min and6.5min, respectively. The thermostability of β-CGTase increased gradually with increasedconcentration of the enzyme. The function of β-CGTase did not required the metal cofactor.However, the cyclization activity of β-CGTase was inhibited by1mM Li+, Mg2+, Zn2+orHg2+, while the enzyme could be activated evidently by1mM Ca2+or Ba2+, increasing10.4%or15.5%, respectively. During the whole cyclization reaction, β-cyclodextrin was the mainproduct. The kinetics of the β-CGTase catalyzed cyclization reaction could not be describedby the Michaelis-Menten equation with corn starch, potato starch or cassava starch as thesubstrate. However, the kinetics of cyclization reaction could be fairly well described by theMichaelis-Menten equation while used soluble starch or maltodextrins (DE5,15,25) as thesubstrate, respectively.(3) The presence of calcium ions had a marked effect on the cyclization activities andthermostabilities of CGTases.1mM Ca2+maximum increased the-cyclodextrin-formingactivity of β-CGTase from B. circulans STB01, increasing of10.4%compared to the absenceof Ca2+. In addition, α-cyclodextrin-forming activity of α-CGTase from Paenibacillusmacerans JFB05-01was increased best in the presence of5mM Ca2+, increasing of nearly15%compared to the absence of Ca2+. However, the effects of Ca2+on the thermostabilities of α-and β-CGTases showed distinctly different. It was noticeable that the increase was muchlower for the α-CGTase compared to the β-CGTase. In addition, the thermostability of thesetwo CGTases showed different response to the calcium ion concentration.0.5mM Ca2+increased the thermostability of α-CGTase maximally, with the half-life prolonged5.5-fold at60oC; while after Ca2+was added to β-CGTase solution to a final concentration of5mMfollowed by incubation for120min at60oC, residual activity of β-CGTase was88.3%, whichwas much higher than that without Ca2+. The results of Nano DSC suggested that anirreversible step took place during denaturation of the β-CGTase. Irreversible denaturation ofthe enzyme was suggested to involve the classical two steps, which could be described by theTwoStateScaled model.0.5~10mM Ca2+increased the Tmand Cpof β-CGTase by1.5~2oCand2.8%~27.6%, respectively.Comparatively analyzed the crystal structure models, the β-CGTase had an additionalcalcium-binding site CaIII than the α-CGTase. Sequence alignment with CaIII of β-CGTaserevealed that the corresponding residues in α-CGTase were Asp and Lys, respectively. Thus,Ala315and Asp577of β-CGTase were replaced by Asp and Lys, respectively, to constructmutants A315D and D577K. The thermostability of mutants A315D and D577K wasimproved significantly compared to the wild type, resulting in80%and130%increases in thehalf-life at60oC, respectively. Further investigation the effects of calcium ions on thethermostability of A315D and D577K revealed that calcium ions improved the thermostabilityof the both mutants, but the overall effects of different concentrations of Ca2+on the thermostability of the D577K mutant were decreased in comparison to mutant A315D. ForA315D, the thermostable effect of Ca2+decreased from a maximum at5to1.5mM incomparison to the wild-type enzyme;86.3%of the β-cyclodextrin-forming activity remainedafter120min of incubation at60°C. However, the optimum concentration of Ca2+forthermostability of the D577K mutant was0.5mM, which retained56.3%of the originalactivity after incubation at60oC for120min. These results suggested that the contribution ofCa2+to CGTase thermostability was closely related to CaIII.(4) Saturation mutagenesis at Asp577had varying influence on the thermostability ofβ-CGTase. Replacements with polar hydrophilic or charged side-chain amino acids had betterthermostability than those of nonpolar hydrophobic side-chains.Correlation analysis of t1/2at55oC and60oC showed that the wild-type and mutantsclustered into three distinct groups. The first group, which contains the wild-type protein, hadthermostabilities very similar to those of the wild-type protein. This group contains, inaddition to the wild-type, the mutants D577I, D577W, D577A, D577L, D577V, D577C andD577S. The second group of mutants included D577E, D577F, D577Q, D577M and D577H.These mutants displayed moderately increased (by36%to45%) half-lives at55oC and60oC.The final group of mutants, which included D577P, D577R, D577T, D577K, D577N, D577Yand D577G, displayed a significant increase in thermostability, with t1/2at60oC prolonged2.1to2.4-fold, and mutant D577G displayed the greatest enhancement.Mechanism analysis illustrated that residue Asp577is located in the periphery of domainD, closely adjacent to domain A. Thus, polar hydrophilic and charged side-chains mightenhance the hydrogen bond, electrostatic interaction or ionic interactions with the nearbyamino acid residues, which could increased the stabity of enzyme structure, and thenimproved the thermostability.(5) Polyethylene glycols (PEGs) with different molecular weights could activate andstabilize tht β-CGTase, but to different degrees. The most significant increase (about20%) inβ-cyclodextrin-forming activity was achieved by adding10%~15%PEG400. PEGs with lowmolecular weights (PEG400and PEG1000) had better enhancement effects on the enzymethermostability than the other PEGs with higher molecular weights. The thermostabilityincreased gradually with increased concentrations of PEG1000. A maximum was observed at15%PEG1000, which increased the half-life of β-CGTase6.5-fold at60oC.Furthermore,0.05%nanosilica sol leads to further increase in PEG1000-enhancedthermostability of β-CGTase. With the simultaneous addition of10%PEG1000and0.05%nanosilica into the enzyme solution, which was allowed to incubate for60min at60oC,61.3%of β-cyclodextrin-forming activity could be retained, which was31.6%higher than that withonly15%PEG1000added. However, addition of nanosilica sol alone had almost no effect onthe thermostability of β-CGTase. Mechanism analysis revealed that PEG increased the surface hydrophobicity of theenzyme molecular, helped protect the tertiary and secondary structure of β-CGTase,respectively. Silica nanoparticles helped PEG1000form a gel network to prevent the enzymefrom aggregating, and then enhanced the enzyme thermostability.