Improvement of enzyme activity, stability and selectivity via immobilization techniques

Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.

Modifying enzyme activity and selectivity by immobilization.

International Technology Roadmap for Semiconductors 2003の要求清浄度について － シリコンウエハ表面と雰囲気環境に要求される清浄度, 分析方法の現状について －

▪ Abstract Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond–forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an α/β hydrolase fold, a catalytic ...

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Bacterial Biocatalysts: Molecular Biology, Three-Dimensional Structures, and Biotechnological Applications of Lipases

Microbial lipases form versatile tools for biotechnology

LIPASES are hydrolytic enzymes which break down triacylglycerides into free fatty acids and glycerols. They have been classified as serine hydrolases owing to their inhibition by diethyl p-nitrophenyl phosphate1. Lipase activity is greatly increased at the lipid-water interface2,3, a phenomenon known as interfacial activation. X-ray analysis has revealed the atomic structures of two triacylglycerol lipases, unrelated in sequence: the human pancreatic lipase (hPL)4, and an enzyme isolated from the fungus Rhizomucor (formerly Mucor) miehei5 (RmL). In both enzymes the active centres contain structurally analogous Asp-His-Ser triads (characteristic of serine proteinases), which are buried completely beneath a short helical segment, or 'lid'. Here we present the crystal structure (at 3 A resolution) of a complex of R. miehei lipase with n-hexylphosphonate ethyl ester in which the enzyme's active site is exposed by the movement of the helical lid. This movement also increases the nonpolarity of the surface surrounding the catalytic site. We propose that the structure of the enzyme in this complex is equivalent to the activated state generated by the oil–water interface.

A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex

Typically pancreatic lipases are characterized by the following properties: (1) they are activated by lipid/water interfaces (interfacial activation), (2) they are inhibited by bile salts but reactivated by colipase (a small activator protein), and (3) they do not hydrolyze significantly phospholipids A cDNA clone encoding a guinea pig pancreatic (phospho)lipase (GPL) has been sequenced and expressed The enzyme (recombinant as well as native) differs from other pancreatic lipases in that (1) it is not interfacially activated, (2) its activity is unaffected by the presence of bile salts and/or colipase using tributyrin as substrate, and (3) it exhibits equally phospholipase A1 and lipase activities The amino acid sequence of GPL is highly homologous to that of other known pancreatic lipases, with the exception of a deletion in the so-called lid domain that regulates access to the active centers of other lipases We propose that this deletion is directly responsible for the anomalous behavior of this enzyme Thus GPL challenges the classical distinction between lipases, esterases, and phospholipases

A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase

The catalytic triad of the neutral lipase from Humicolalanuginosa is buried by a short helix under aqueous conditionsrendering the enzyme inactive. Upon adsorption to a lipidsubstrate interface this helix is displaced, thereby exposingthe active site (interfacial activation). By covalently linkinginhibitors to the active serine, it is possible to crystallize theenzyme in an interfadally activated state. Two such structuresare reported here which mimic the tetrahedral transitionstates of lipolysis. To date, no crystal structures of alipase—triglyceride complex exist for this enzyme. Therefore,possible interactions between this lipase and its substrate havebeen analysed through molecular modelling.Key words: crystal structure/fungal lipase/inhibitors/substratemodellingIntroductionThe structure of the triacylglyceride lipase isolated fromthe filamentous fungus Humicola lanuginosa (HLL) wassolved several years ago in York at 3.5 A resolution, and sub-sequently refined to 2.5 A resolution (Lawson et al., 1994;A.M.Brzozowski et al., manuscript in preparation). Morerecently, another crystal form of this enzyme has enabled thestructure to be refined to 1.8 A resolution in Edmonton, Canada(Derewenda et al., 1994). Its structure is closely similar to thatof Rhizomucor miehei lipase (RML; Brady et al., 1990),comprising a single, roughly spherical domain containing a centraleight-stranded, predominantly parallel /3-pleated sheet, and fiveinterconnecting a-helices. The active site contains a Ser-His-Aspcatalytic triad closely reminiscent of that seen in the serineproteases. Under aqueous conditions, access to this catalytic triadis blocked by a short helical 'lid'. In common with the RMLstructure, there is a tryptophan residue (Trp89) in this helix whichlies directly over the active serine.When complexed with substrate analogues or serine proteaseinhibitors, the lid helix is displaced and the active site is exposed(Figure 1). This conformational change is thought to be thestructural basis for the phenomenon of interfacial activation seenin all classical lipases (Desnuelle et al., 1960). This changegenerates a large hydrophobic surface, which includes the activesite, and is stabilized by adsorption to a hydrophobic interface(Lawson et al., 1992). Two such structures, which are thoughtto mimic the tetrahedral transition states of the lipolyticmechanism, are reported here. Using the results of these studiesas a starting point, we have been able to model substrate bindingto the enzyme.HLL is termed a 1,3-specific lipase, as it preferentiallyhydrolyses fatty acids from the snl and sn3 positions of thetriglyceride molecule. Moreover, although chemically identicalin a triglyceride molecule comprising only one fatty acid type,hydrolysis at the snl position is preferred in this lipase (Rogalskaet al., 1993). This has been attributed to the stereochemical non-equivalence of these two groups.Materials and methodsPreparation of crystalsAll protein samples were provided in recombinant purified formby Novo-Nordisk A/S (Copenhagen) and crystallized using thehanging drop technique. For preparation of crystals of inhibitorcomplexes, the protein was incubated with the inhibitor prior tocrystallization. The phosphonate inhibitor was also supplied byNovo-Nordisk.Data collection and processingX-ray data were collected using either a Nicolet Xentronics areadetector or a Rigaku R-AXIS II image plate on Rigaku RU200rotating anode generators run at 5 kW. CuKa radiation (1.542A) was provided through a graphite monochromator. Dataprocessing for the area detector data was performed with theXENGEN package (Nicolet Instrument Co., 1987) and with theMOSFLM package (Leslie et al., 1986) for image plate data.Data reduction was carried out using programs from die CCP4program suite (SERC Daresbury Laboratory, 1979).Structure solution, electron density fitting and refinementThe two crystal structures were solved by the molecularreplacement technique (Rossmann and Blow, 1962) using the freeenzyme as a search model (A.M.Brzozowski et al., manuscriptin preparation). The MERLOT (Fitzgerald, 1988) or AMORE(Navaza, 1990) packages were used. Model building wasperformed using the program FRODO (Jones, 1978) on an Evansand Sutherland ESV50 graphics workstation with electron densitymaps calculated using model phases with the coefficients [2F

Probing the nature of substrate binding in Humicola lanuginosa lipase through X-ray crystallography and intuitive modelling

Lipases that break down triglycerides to monoglycerides and glycerol are characterised by low or no activity in water; in the presence of an oil/water interface, however, their activity increases markedly. The structural and chemical basis for this phenomenon, referred to as interfacial activation, has been revealed by the crystal structures of a fungal lipase and a human pancreatic lipase which evidently have a divergent evolutionary history. These studies reveal that: (1) In both enzymes the catalytic sidechains are Asp:His:Ser, the same as occur in the serine proteases. The active atoms on this catalytic triad have essentially identical stereochemistry in the serine proteases and in these two lipases. The amino acids themselves, however, have quite different conformations and orientations. (2) In both enzymes the catalytic groups are buried and inaccessible to the surrounding solvent. Burial in these two lipases is brought about by a small stretch of helix (the lid) which sits over the active site. (3) In both enzymes this helical lid presents non-polar sidechains over the catalytic group, and polar sidechains to the enzyme surface. Although the 'lids' are very similar in construction in the two enzymes, they belong to very different parts of the polypeptide chain. (4) Although the amino acid sequences have no identity (except at the active serine) the two enzymes show a similar architectural framework consisting of a central five-stranded parallel beta sheet structure. The catalytic groups decorate this beta sheet structure in a strikingly similar way though there are also some significant differences. The crystal structure of the complex between the fungal enzyme and a substrate analogue demonstrates how the helical lid is displaced to reveal the active site. The movement of the lid also greatly enlarges the non-polar surface at the active surfaces and buries previously exposed polar residues. The movement of the lid also helps to create the appropriate movement at the oxyanion hole. It is possible to define the stereochemistry at the active site and to identify the positioning of the fatty acid and the glycerol moieties.

Structural and evolutionary relationships in lipase mechanism and activation.

This paper explores the potential for transistor level fault tolerance on a new Programmable Analogue and Digital Array (PAnDA) architecture1. In particular, this architecture features Combinatorial Configurable Analogue Blocks (CCABs) that can implement a number of combinatorial functions similar to FPGAs. In addition, PAnDA allows one to reconfigure features of the underlying analogue layer. In PAnDA-EINS, the functions that the CCAB can implement are predefined through the use of a routing block. This paper is a study of whether removing this routing block and allowing direct control of the transistors provides benefits for fault tolerance. Experiments are conducted in two stages. In the first stage, a logic function is evolved on a CCAB and then optimised using a GA. A fault is then injected into the substrate, breaking the logic function. The second stage of the experiment consists of evolving the logic function again on the faulty substrate. The results of these experiments show that the removal of the routing block from the CCAB is beneficial for fault tolerance.

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David M. R. Lawson

Papers

A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex

A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase

Probing the nature of substrate binding in Humicola lanuginosa lipase through X-ray crystallography and intuitive modelling

Structural and evolutionary relationships in lipase mechanism and activation.

Overcoming faults using evolution on the PAnDA architecture