Jesper Vind

Phospholipases and their industrial applications

Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A1, A2, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions—white biotechnology is a strong alternative.

Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase

The mechanism of allosteric activation of α‐amylase by chloride has been studied through structural and kinetic experiments focusing on the chloride‐dependent N298S variant of human pancreatic α‐amylase (HPA) and a chloride‐independent TAKA‐amylase. Kinetic analysis of the HPA variant clearly demonstrates the pronounced activating effect of chloride ion binding on reaction rates and its effect on the pH‐dependence of catalysis. Structural alterations observed in the N298S variant upon chloride ion binding suggest that the chloride ion plays a variety of roles that serve to promote catalysis. One of these is having a strong influence on the positioning of the acid/base catalyst residue E233. Absence of chloride ion results in multiple conformations for this residue and unexpected enzymatic products. Chloride ion and N298 also appear to stabilize a helical region of polypeptide chain from which projects the flexible substrate binding loop unique to chloride‐dependent α‐amylases. This structural feature also serves to properly orient the catalytically essential residue D300. Comparative analyses show that the chloride‐independent α‐amylases compensate for the absence of bound chloride by substituting a hydrophobic core, altering the manner in which substrate interactions are made and shifting the placement of N298. These evolutionary differences presumably arise in response to alternative operating environments or the advantage gained in a particular product profile. Attempts to engineer chloride‐dependence into the chloride‐independent TAKA‐amylase point out the complexity of this system, and the fact that a multitude of factors play a role in binding chloride ion in the chloride‐dependent α‐amylases.

Studies on ferulic acid esterase activity in fungal lipases and cutinases

In this study we have tested a number of lipases, lipase variants and cutinases for ferulic acid esterase activity, using ferulic acid ethyl ester as substrate. It was shown that Thermomyces lanuginosa lipase (TLL), Candida antartica lipase A, Candida antartica lipase B, Rhizomucor miehei lipase and Fusarium oxysporum lipase have no significant ferulic acid esterase activity. Thirteen variants of TLL were constructed based on a model of Aspergillus niger ferulic acid esterase A (FAE-A). Activity assay using ferulic acid ethyl ester as substrate gave, for FAE-A, 112 U/mg=112 μmol ferulic acid released per min per mg enzyme. Two of the variants of TLL had significant ferulic acid esterase activity, TLLv1 (7 U/mg) and TLLv10 (20 U/mg). Both these variants contain the mutation F113Y that seems to be essential for ferulic acid esterase activity. In addition to lipase activity, three cutinases showed ferulic acid esterase activity, Aspergillus oryzae cutinase (5 U/mg), Fusarium solani pisi cutinase (13 U/mg), Humicola insolence cutinase (20 U/mg).

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.

Impact of the physical and chemical environment on the molecular structure of Coprinus cinereus peroxidase.

The structure of the peroxidase from Coprinus cinereus (CiP) has been determined in three different space groups and crystalline environments. Two of these are of the recombinant glycosylated form (rCiP), which crystallized in space groups P2(1)2(1)2(1) and C2. The third crystal form was obtained from a variant of CiP in which the glycosylation sites have been removed (rCiPON). It crystallizes in space group P2(1) with beta approximately 90 degrees; the structure was determined from room-temperature data and low-temperature data obtained from twinned crystals. Two independent molecules of CiP related by non-crystallographic symmetry are contained in the three crystal forms. The packing in the two structures of the glycosylated form of rCiP is closely related, but differs from the packing in the unglycosylated rCiPON. A database search based on small-molecule porphinato iron (III) complexes has been performed and related to observations of the spin states and coordination numbers of the iron ion. The room-temperature structures of CiP and one structure of the almost identical peroxidase from Arthromyces ramosus (ARP) have been used to identify 66 conserved water molecules and to assign a structural role to most of them.

Glycosylation of Thermomyces lanuginosa lipase enhances surface binding towards phospholipids, but does not significantly influence the catalytic activity

Abstract Binding properties of the native Thermomyces lanuginosa lipase (Tll), the inactive mutant of Tll (S146A; active Ser146 mutated to Ala), and the non-glycosylated mutant of Tll (N33Q) were determined using fluorescence spectroscopy. Tll, S146A mutant and N33Q mutant show significant different binding behavior to phosphatidylcholine (PC) and phosphatidylglycerol (PG) liposomes. Generally, weaker association of lipase molecules is observed to PC liposomes than to PG liposomes. Strong lipase–lipid interactions are observed for the S146A mutant, which is less pronounced for Tll and the N33Q variant. Addition of fatty acid to PG liposomes reduces significantly the binding affinity of the lipases. This effect is less pronounced in fatty acid/PC liposomes. Although the catalytic activity of the N33Q mutant is comparable to Tll, the non-glycosylated variant shows generally lower binding affinity to PC or PG matrix than Tll. Addition of the substrate analog benzene boronic acid (BBA) increases the binding affinity of the S146A and N33Q mutants, while only small changes are observed for Tll suggesting that the dynamics of the active site lid influences the binding affinity and that the flexibility of the loop region 33–48 might contribute to the activation of the lipase.

Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function.

Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.

Advanced synthesis of conductive polyaniline using laccase as biocatalyst

Polyaniline is a conductive polymer with distinctive optical and electrical properties. Its enzymatic synthesis is an environmentally friendly alternative to the use of harsh oxidants and extremely acidic conditions. 7D5L, a high-redox potential laccase developed in our lab, is the biocatalyst of choice for the synthesis of green polyaniline (emeraldine salt) due to its superior ability to oxidize aniline and kinetic stability at the required polymerization conditions (pH 3 and presence of anionic surfactants) as compared with other fungal laccases. Doses as low as 7.6 nM of 7D5L catalyze the polymerization of 15 mM aniline (in 24 h, room temperature, 7% yield) in the presence of different anionic surfactants used as doping templates to provide linear and water-soluble polymers. Aniline polymerization was monitored by the increase of the polaron absorption band at 800 nm (typical for emeraldine salt). Best polymerization results were obtained with 5 mM sodium dodecylbenzenesulfonate (SDBS) as template. At fixed conditions (15 mM aniline and 5mM SDBS), polymerization rates obtained with 7D5L were 2.5-fold the rates obtained with commercial Trametes villosa laccase. Moreover, polyaniline yield was notably boosted to 75% by rising 7D5L amount to 0.15 μM, obtaining 1g of green polyaniline in 1L-reaction volume. The green polymer obtained with the selected system (7D5L/SDBS) holds excellent electrochemical and electro-conductive properties displayed in water-dispersible nanofibers, which is advantageous for the nanomaterial to be readily cast into uniform films for different applications.

Promoting protein self-association in non-glycosylated Thermomyces lanuginosus lipase based on crystal lattice contacts.

We have used the crystal structure of Thermomyces lanuginosus lipase (TlL) to identify and strengthen potential protein-protein interaction sites in solution. As wildtype we used a deglycosylated mutant of TlL (N33Q). We designed a number of TlL mutants to promote interactions via interfaces detected in the crystal-lattice structure, through strengthening of hydrophobic, polar or electrostatic contacts or truncation of sterically blocking residues. We identify a mutant predicted to lead to increased interfacial hydrophobic contacts (N92F) that shows markedly increased self-association properties on native gradient gels. While wildtype TlL mainly forms monomer and <5% dimers, N92F forms stable trimers and dimers according to Size-Exclusion Chromatography and Small-Angle X-ray Scattering. These oligomers account for ~25% of the population and their enzymatic activity is comparable to that of the monomer. Self-association stabilizes TlL against thermal denaturation. Furthermore, the trimer is stable to dilution and requires high concentrations (>2M) of urea to dissociate. We conclude that crystal lattice contacts are a good starting point for design strategies to promote protein self-association.

Artificial Evolution of Fungal Proteins

Numerous techniques have been developed over the last two decades to change characteristics of proteins, and with these techniques it has become feasible to customize proteins for specific commercial and environmental applications. For instance, a fungal lipase from Thermomyces lanuginosus has been improved to increase its performance in detergent solutions (Borch et al., 1998). The same lipase has been changed into a phospholipase that can be applied in bread making to generate emulsifiers from the lipids present in the dough, eliminating the need to add emulsifiers (Bojsen et al., 1998). This chapter will provide a general overview of the advances within artificial molecular evolution. It will focus on the in vitro and in vivo procedures that have been applied to fungal proteins, some techniques having been developed specifically for filamentous fungi. Finally, thoughts on the future direction of artificial evolution of fungal proteins will be offered.