Study Of The Specific Site Covalent Immobilization And Catalytic Promiscuity Of Acyl-peptide Releasing Enzyme ST0779

Enzymes have been widely used in the field of chemical, food, pharmaceutical,environmental protection and detection as efficient biocatalysts.But it is unstable andeasily denatured when exposed to unfavorable condition such as temperature, pH and soon. Immobilized enzyme can availably overcome these applied limits and allow reusingenzyme to catalyze. Enzyme immobilization has been a hotspot in the field of enzymeehgineering.Currently immobilization method can be divided into non-specific and specific. Inrecently years, the development and applications of specific immobilization haveundergone significant advances. This straightforward method of protein immobilizationis through the use of the rational design and renovation fusing the functional groups,which react specifically with an immobilized metal ion, inhibitor or chemical groups.This method aimed at increasing the activity, stability or catalytic specificity of targetenzymes.In this paper, we focused on the specific site covalent immobilization. Through therational design and renovation, the enzyme wasintroduced the aldehyde functionalgroup forspecific site covalent immobilization on the carrier with the amino group. Thismethod could not only screen the rational design of enzyme to immobilized, and alsostably connect between the enzyme and the carrier by formation of covalent bondformation. Formylglycine generating enzyme (FGE)could recognize highly amino acidconserved sequence LCXPXR sequence in sulfatase and catalyze the cysteine in thesequence to convert the aldehyde containing a formyl glycine (FGly).These sixconserved sequences were introduced to genetically engineered protein. To obtain theprotein with aldehyde group, we applied this FGE to catalyze the fusing of theconserved sequence protein by the FGE.As commercial plasmid, pET-28a contains two His-tags, which makes protein purification easier. So we chose pET-28a as a model plasmid. His-tag located in N andC terminal of the multiple cloning sites. His-tags were replaced with an aldehyde grouplabel (aldehyde-tag) for specific site immobilization. We constructed four types ofexpression vectors: pET-28aDH without His-tag, pET-28aNQ with aaldehyde tag at Nterminal, pET-28aCQ with a aldehyde tag at C terminal and pET-28aDQ with doublealdehyde tags at the both terminals. We choose four kinds of enzymes as model enzymeby introducing aldehyde tag at different location for specific site immobilization,respectively thermophilic esterase APE1547, thermophilic acyl-peptide releasingenzyme ST0779, thermophilic dehalogenase ST2570and Lipase LipA. We measuredthe immobilized enzyme efficiency, stability and recycling times of these for researchaldehyde tag to specific site immobilization.Catalytic promiscuity is one of the most exciting discoveries in enzymology fieldafter non-/micro-aqueous enzymology, which is of particular interest for in vitrobiosynthesis and organic synthesis by possibly improving existing catalysis or discoveryof novel synthesis pathways that are currently not available.In the efforts towards theidentification and engineering of promiscuous enzymes to catalyze aldol additionreactions, several successful cases have been reported; most of which belong to/βhydrolase family.Acyl-peptide releasing enzyme ST0779belongs to/β hydrolasefamily.Noticing many of hydrolases of which ST0779belongs to, having promiscuousactivity, we thus assumed that ST0779might have promiscuous activity as well. In thiswork, ST0779was used to catalysed aldol reaction, which showed a superior activity atelevated temperature. The turnover numbers kcat (s-1) of this thermostable enzyme at55°C is7.78-fold higher than that of PPL at its optimum temperature37°C; and themolecular catalytic efficiency kcat/Km (M-1s-1) added up to140times higher than PPL.The fluorescence quenching analysis depicted that the binding (dis-association)constants of PPL are significantly higher than those of ST0779; and their number ofbinding sites showed opposite temperature-dependency. The increase in the number ofbinding sites of ST0779with increasing temperature demonstrated the increasing affinity to substrates, which might explain its markable enhanced activity at elevatedtemperature. Thermodynamic parameters estimated by fluorescence quenching analysisunrevealed distinctly different substrate-binding modes between PPL and ST0779: thegoverning binding interaction between PPL and substrates is hydrophobic force; whilethe dominating substrate-binding forces for ST0779are van der Waals and H-bondsinteractions. A plausible mechanism in ST0779catalyzed aldol reaction was proposedbased on kinetic study, spectroscopic analysis and molecular stereo structure simulation.This work represents a successful example to identify a new enzyme for catalyticpromiscuity, which demonstrated the huge potential to discover and exploit novelbiocatalyst from thermophile microorganism sources.