Biochimica et Biophysica Acta 1543 (2000) 223^238

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Review

Lipase protein engineering

Allan Svendsen *
Enzyme Design, Novo Nordisk A/S, Novo Alle, 2880 Bagsvrd, Denmark Received 3 March 2000; received in revised form 11 September 2000; accepted 28 September 2000 Keywords: Lipase; Protein; Engineering ; Enzyme

1. Introduction Lipases are enzymes, which catalyse the hydrolysis or formation of lipids. The lipase discussed in the present review has mainly the triacylglycerol activity and is classied in the EC 3.1.1.3 group. The reaction is shown by the following equation: TriglycerideHGlycerol Fattyacids The lipases are a versatile group of enzymes and often express other activities like, e.g., phospholipase, lysophospholipase, cholesterol esterase, cutinase, amidase and other esterase type of activities [1,2,27]. Generally lipases have preference for the substrate type, whether it is a triglyceride or a diglyceride, and therefore have diglyceride and monoglyceride as products, rather than the glycerol and fatty acids alone. The regioselectivity is often rather high for the positions sn1 and sn3 and less frequently the sn2 is degraded (see Fig. 1) [1,3,4]. The lipase enzymes consist of a large family showing the same overall structural fold [5,28], but which has a versatility of loop structures in contact with the substrate, and exhibits versatile substrate specicities. Another denition of lipases often used is lipolytic enzymes, which are capable of hydrolysing lipid substrates, thus including phospholipases, cutinases or enzymes

hydrolysing ester substrates of lipid character. Protein engineering of selected other lipases, hydrolases or enzymes and proteins having homologous X-ray structures is also occasionally included in this review. For general protein engineering references including lipases see [30,35]. Lipases are enzymes with general interest within many industrial applications. Lipases are used within the industry, e.g., detergents, oil and fats, baking, organic synthesis, hard surface cleaning, leather industry and paper industry [1,2,6^9,41,58]. Protein engineering of triacylglycerol lipases has been done since the mid-1980s. The rst example on protein engineering of a lipase is the work on the Pseudomonas mendocina lipase [10]. The early work was done based only on sequence information. When the rst structures became available in the late 1980s the protein engineering interest increased dramatically. A European funded project, as well as

* Corresponding author. Fax: +45-4498-0246; E-mail: asv@research.novo.dk

Fig. 1. The triacylglyceride type of lipid presented using the sn designation for positions of the glycerol moiety.

0167-4838 / 00 / $ ^ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 2 3 9 - 9

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many academic and industrial laboratories around the world, focused on solving new lipase structures of the enzymes Rhizomucor miehei lipase [11], Humicola lanuginosa lipase [12] (now Thermomyces lanuginosa lipase), Pseudomonas glumae lipase [13], Fusarium solani pisi cutinase [14], Candida antarctica B lipase [15] and Candida rugosa lipase [16] (or Candida cylindracea lipase), and most of these enzymes became targets for protein engineering. Now more than 12 X-ray structures of lipases are available in the protein database (PDB), of which 10 are microbial. Many of the lipases are solved in both a closed and an open conformation, i.e., with the lid or lids displaced from the active site. The Rhizomucor miehei lipase [11] and Human pancreatic lipase [17] X-ray structures was the rst to be published. The Rhizomucor miehei lipase X-ray structure was found to have an active site triad as in the proteases [11]. Later by solving the X-ray structure in a form inhibited by a phosphonate inhibitor [18], which covalently binds to the active site Ser, it was revealed that the lid was displaced from the active site by a hinge bending movement [20], creating an increased hydrophobic surface [21]. Other lipases have also been crystallised with a number of dierent substrate
Table 1 Selected lipase X-ray structures listed with references Lipase Open

analogous like the Candida antarctica B lipase [22], Human pancreatic lipase [23], Pseudomonas sp. lipase [24], Fusarium solani pisi cutinase [25] and Candida rugosa lipase [19,26]. A list of selected solved lipase structures is given in Table 1. From many of these structures lipases with homologous sequences can be model build, using adequate programs as, e.g., Homology or Modeller from MSI [27], or using threading programs [28]. Protein engineering of other lipases has also been performed, e.g., of the lipoprotein lipases [29] and the phospholipases, especially the phospholipase A2 (PLA2 ) [30]. Within the mammalian lipases many point mutations have been isolated from various sources including humans. These mutations are responsible for several diseases related to the lipase function [31]. Surface charge variants in Humicola lanuginosa lipase have been addressing identication of epitopes of the wild-type enzyme [43]. The overall structure of the triacylglycerol lipases can be described as a structure with a central L-sheet with the active serine placed in a loop (see Fig. 2), termed the catalytic elbow. Above the serine a hydrophobic cleft are present or formed after activation of the enzyme [32,33]. The hydrophobic cleft is an elon-

Closed

Others

Thermomyces lanuginosa (Humicola lanuginosa) Rhizomucor miehei 4TGL, 5TGL

Penicillium camenbertii Rhizopus oryzea (niveus, arhenius, delemar) Pseudomonas glumae (chromabacterium viscosum) Pseudomonas cepacia Fusarium solani pisi

1TIB 1TGL, 2TGL, 3TGL 1TIA 1TIC 1TAH, 1QGE, 1CVL 1OIL, 2LIP, 3LIP (without inh.) (incl. 4LIP, 5LIP Inhibitor)

Candida antarctica B Candida rugosa (Candida cylindracea)

Geotrichum candidum Human pancreatic lipase

^ ^ 1CRL (without inhibitor) Complexes: 1LPN, 1LPS, 1LPO, 1LPM, 1TRH 1THG 1LPB

1CUS (wt), Engineered: 1CUA-J, 1CUU-Z, 1OXM, 1XZA-I, Complexes: !XZJ-M 1LBT, 1LBS, 1TCB, 1TCC 1LPP

Complexed with colipase and phospholipid: 1LPA

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Fig. 2. Lipase overall structure presented using the Humicola lanuginosa lipase crystal structure [122] and presented in Molscript representation [114]. The L-sheet is shown in blue, surrounded by some helices (yellow), and the active serine site residue in red sticks, and the lid shown in red. Both the open and the closed conformation are shown superimposed.

gated pocket suitable for acyl moieties to t into. The activation, which is often necessary for the lipase enzyme, is a movement of a lid or several lids. This process is a part of the activation of activity, often referred to as interfacial activation [34], which takes place above the critical micellar concentration of the substrate. In Humicola lanuginosa lipase the opening of the lid can be described as a hinge bending motion of a helical lid. For other lipases the activation is more complex, e.g., Human pancreatic lipase [23] and Candida rugosa lipase [36], having both more than one lid or ap. In addition, activation is only present for certain substrates, e.g., Fusarium solani pisi cutinase, has a small structural change associated with long chained acyl substrates [36], whereas there is no or little change of structure upon binding of phosphonate inhibitor for the more esterase like enzyme of Candida antarctica B lipase [22]. The lipases belong to the K/L hydrolase family [5]. The K/L hydrolase family contains enzymes within lipases, esterases, proteases, peroxidases and lyases and others [5,37]. Not all lipases belong to the same structural families, as, e.g., the PLA2 , PLA-D, and some lysophospholipases, having very distant structures compared to the triacylglycerol lipases. The hydrophobic surface area is placed centrally in the lipid contact zone. This lipid contact zone has been addressed by protein engineering. To locate the lipid contact zone, cysteine residues have been engineered into the surface of PLA2 [38]. Binding of spin labels to the cysteines and measuring the

Fig. 3. Lipid contact zone of the Humicola lanuginosa lipase presented in cpk model in (a) open form and (b) closed form. The hydrophobic residues are coloured white, hydrophilic residues yellow, positively charged residues blue and negatively charged residues red.

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Fig. 4. Ribbon structures of Pseudomonas glumae lipase (a), Candida rugosa lipase (b), Candida antarctica B lipase (c), Humicola lanuginosa lipase (d), Fusarium solani pisi cutinase (e), and Human pancreatic lipase (f). The tentatively placed lid or lids are coloured white.

ESR signal perturbation on binding to the lipid surface, indicates a special orientation of binding to the macroscopic substrate surface. The suggested lipid contact zone of the Humicola lanuginosa lipase is shown in Fig. 3. In the Humicola lanuginosa lipase the lipid contact zone contains several big hydrophobic residues like Phe, Trp, Ile, Leu and Tyr. Some of the Phe and Ile residues are suggested to function as lipid anchors, and thus probably penetrates into the hydrophobic part of the lipid surface. The lid is also a central part of the lipid contact zone of most triacylglycerol lipases. The displacement of the lid increases the hydrophobic surface area of the lipases dramatically within the lipid contact zone area. A number of lipase structures with their lid or lids highlighted are shown in Fig. 4. The lipases can be grouped into subfamilies by sequence homology analysis [39^41] based on sequence homology, thus dividing them into two main families, the mammalian and the microbial lipase family. Within microbial lipases, several families have now been found, the bacterial lipases, containing the Staphylococcus lipase family, Pseudomonas lipase family, Bacillus lipases and others, and the fungal lipases with the Rhizomucor miehei lipase fam-

Table 2 A list of selected protein engineering topics and ideas in selected lipases Lipase Humicola lanuginosa Variants addressing Ref. [103] [81,103] [81,104] [77] [59] [54,66,67] [57] [106] [70] [105] [75,76] [75,76] [70] [69,70] [108,109] [49] [49] [63] [71] [87] [85] [47] Surface charge Detergents Localised random Protease stability Activity Rhizopus delemar Chain length Pseudomonas mendocina Perhydrolysis Detergents Pseudomonas glumae Protease stability Detergents Pseudomonas aeruginosa Enantiomer selectivity Directed evolution Fusarium solani pisi Anionic sensitivity Chain length Crystal structure Candida antarctica B Specicity Oxidation stability Staphylococcus hyicus Phospholipase activity Geotrichum candidum Specicity Lipoprotein lipase Flap deletions Human pancreatic lipase Flap deletions Lysosomal acid lipase Cholesterol esterase activity

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ily, Candida rugosa lipase family [39,41,42], and other subfamilies. Within microbial lipases the fungal family of Rhizomucor miehei family and the bacterial family of Pseudomonas sp., as well as the Fusarium solani pisi cutinase have been devoted most protein engineering interest. See Table 2 for overview of selection of the lipases and protein engineering issues discussed in the following sections. The following topics of lipase protein engineering will be discussed: variants addressing active site, variants addressing activity (including specic activity, substrate type specicity and chain length specicity), variants addressing stability (including temperature stability, protease stability and oxidation stability), variants addressing lid function, variants addressing macroscopic substrate interaction, variants addressing calcium binding, variants addressing detergent use and surfactant compatibility, and variants addressing X-ray structure and dynamics. 2. Variants addressing active site Variants in the cutinase type lipase from Pseudomonas mendocina focused on identifying the active site residues and changes in the nearest residues in the sequence. Using this idea the active site Ser and

Fig. 5. Active site conformation of the Humicola lanuginosa lipase with the diethyl phosphonate inhibitor bound in the active site [122], showing the active site triad with the Asp201, His258 and Ser146. The oxyanion hole is visualised by the Ser83, which has hydrogen-bonding contact to the oxygen of the carbonyl (here oxygen bound to a phosphorus atom). The carbon atoms are in green, oxygen in red, nitrogen in blue and hydrogen in white.

His were suggested, and changes in activity, especially perhydrolysis, was obtained [10]. After obtaining the structures of the rst lipases the active site residues were found to be homologous to the triad of serine proteases (Fig. 5). In Humicola lanuginosa lipase and Rhizomucor miehei lipase the active site Ser was identied by using phosphonate inhibitors [20] and later by site-directed mutagenesis of the SerC Ala. The general problem in identifying the active site residues with certainty is that the mutated enzyme often has a rest activity and that the mutations can destroy activity even though they are not specifically in the active site. However, the method is applicable in most cases. Several authors have addressed the active site triad residues by site-directed mutagenesis, e.g., Candida antarctica B lipase and Candida antarctica A lipase [43], Staphylococcus hyicus [44], pancreatic cholesterol esterase [45], lysosomal acid lipase [46], and hormone sensitive lipase [47]. The method of identifying the active site residues by site-directed mutagenesis and only based on sequence has also been suggested using sequence homology based on structural knowledge [39]. The active site Ser is most often found in the consensus sequence Gly-Xxx-Ser-Xxx-Gly, but in some cases the consensus sequence has been found to be missing in the sequence [49,50]. The His residue is often part of a special sequence pattern [39], and further the active site Asp residue can be found in the triacylglycerol lipases approximately in the mid between the Ser and the His. In some lipase related enzymes different types of arrangements of the active site residues and the oxyanion hole have been found [51] as, e.g., in the Streptomyces scapies esterase where the Asp is `replaced' by a carbonyl oxygen. The catalytic mechanism of a lipase has been addressed thoroughly in Rhizopus oryzea lipase [52] by substitution of many of the residues in the substratebinding region around the active site. The substitutions were made on basis of a model build based on the Rhizomucor miehei lipase X-ray structure. Variants in the positions 28 and 143 of the Rhizopus oryzea lipase nearly destroyed the activity. Both positions are part of a hydrogen bonding network in the active site surroundings. The authors suggested that it demonstrated the importance of keeping the active site dynamics and structure intact. One can also draw the conclusion that it is not always muta-

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Fig. 6. Sequence alignment based on the three-dimensional structures of Rhizomucor miehei lipase (lip_rhimi [115]), Rhizopus oryzea lipase (lip_rhidl [116]) (identical to Rhizopus delemar lipase), Penicilium camembertii lipase (pcmdgl [117]) and Humicola lanuginosa lipase (HLL [118]). The lipid contact zone extracted from Humicola lanuginosa lipase is highlighted in bold in sequence HLL. Also highlighted is residues mentioned in the chapter on specicity and enantioselectivity (bold in sequence lip_rhidl).

tions of the active site residues, which dramatically lowers or destroys the activity of an enzyme. The residue stabilising the carbonyl group of the substrate in the hydrolysis in Rhizopus oryzea lipase in the oxyanion hole is threonine 82. The T82 was substituted for an Ala residue and resulted in a virtually inactive lipase, but a T82S variant, where a hydroxyl group is still present, resulted in a 12% active lipase compared to the wild type. The lower activity of the T82S compared to the wild type could be explained by that the Ser introduced in position 82 were more frequently making hydrogen bonds to the D91, as seen by molecular dynamics simulations. Similar results were obtained for the variants T83A and T83S in Rhizopus delemar lipase [54]. The homologous variant in Fusarium solani pisi cutinase was made, S42A,

in order to analyse the tetrahedral intermediate stability given by the Ser hydroxyl group [53]. The A88 in Rhizopus oryzea corresponding to the W89 in Humicola lanuginosa lipase and A89 in Rhizopus delemar lipase (see Fig. 6) is placed directly on top of the active site serine. The A88W variant in Rhizopus oryzea lipase showed less activity compared to the wild type in accordance with the result found for A89W in Rhizopus delemar lipase [54]. This nding is partly in contrast to the result found for Humicola lanuginosa lipase where substitution of the W89 to F, L, G or E gave lower activity against tributyrin [55]. The dierence is probably due to the local surroundings of the W89 and A89 in the two enzymes. An attempt to probe an alternative triad acid residue by substitution of E265D destroyed activity, whereas

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E265Q has activity corresponding to the Rhizopus oryzea lipase wild type. In Geotrichum candidum lipase the shift of the active site acid to the same conguration as in the Human pancreatic lipase, resulted in an activity 10% lower than the wild type measured on a uorogenic substrate 4-methylumbelliferyl-oleate [56]. 3. Variants addressing activity 3.1. Specic activity Variants made within 6^7 residues from the active site residues in Pseudomonas mendocina cutinase (identied as mentioned above), have in assays been shown to alter the specic activity of perhydrolysis. The ratio of perhydrolysis/trioctanoin hydrolysis was increased by approximately four times [57]. Mutations made in the lipid contact zone of Humicola lanuginosa lipase have been shown to alter the specic activity as well. Normally the specic activity is decreased by mutations, but some special sites increasing the specic activity are possible to identify. The last is especially true for increasing the activity against a non-natural substrate of which the enzyme is not optimised, like, e.g., the perhydrolysis mentioned earlier. In Candida antarctica A lipase the specic activity has been increased by four times based only on sequence alignments by making a

FCW mutation [81]. In Humicola lanuginosa lipase the mutation of the W89 in the lid has been shown to decrease the specic activity towards triglycerides [55]. Analysis of the reaction scheme (see Fig. 7) of the W89 mutant lipases has been addressed and the acylation reaction rate was exclusively altered, suggesting the residue W89 importance for the acylation step [60]. Variants in the position W89 was found to have a positive eect on substrates with bulky acyl chains resulting in higher activity in contrast to the natural glycerol substrates. The activity of the variant E87A in Humicola lanuginosa lipase was found to be almost independent of the substrate type measured on triacetin, tripropionin and tributyrin [60]. Of mutations reported to eect the activity of lipases in the removal of the N-glycosylations or only one glycosylation site, N308, in Human gastric lipase, resulted in only 50% active lipase measured on short and long chained substrates [61], and the removal of a cystine bridge C227/C236 by substitution into Ala lowered the activity down to 13^17% on triolein and tributyrin [62]. 3.2. Substrate type specicity The phospholipase activity from Staphylococcus hyicus lipase has been lowered 12 fold by introduction of the mutation S356V in Staphylococcus hyicus lipase [63]. The Val residue is present in Staphylococcus aureus lipase, which has no phospholipase activ-

Fig. 7. A general reaction scheme for the interaction of a lipase with the substrate is shown. Reaction 1 illustrates the process from soluble enzyme to bound active enzyme. This process could consist of the states of (1) binding, (2) orientation, (3) activation (here lid displacement) and nally (4) getting a monosubstrate into the active site. The rest of the reaction scheme (reaction 2) represents the catalytic reaction including the proper substrate enzyme interaction making the tetrahedral intermediate (Ea*S), the acylation step (Ea*SHEa*Ac+P1), and the deacylation step (Ea*AcHEa*+P2). a, adsorbed enzyme; *, activated.

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ity. The technique used to identify this residue was domain exchange between the two highly homologous lipases. The C-terminal domains were found to be most important for expression of the phospholipase activity. The reverse mutation V357S in Staphylococcus aureus lipase however, does not make the Staphylococcus aureus into a phospholipase. The 356/ 357 position in the two Staphylococcus lipases is placed just next to the active site His. The loss of cholesterol esterase activity of lysosomal acid lipase [47], was found by the substitutions L179P or L336P. The importance of V710 and S563 for activity has been shown for rat hormone-sensitive lipase. The S563A mutation received the activity cholesteryl ester [48]. Reactivation of an inactive form of pancreatic like lipase, PLRP1, has been reported by substitution by homology comparison of V to A and A to P in the vicinity of the active site Ser [64,65]. 3.3. Chain length specicity The chain length specicity has been analysed by protein engineering for the Rhizopus delemar lipase [54,66,67] and the lipase homologous acetyl-cholinesterase from rat brain [68]. The binding groove for an

Fig. 8. Acyl binding cleft of Rhizomucor miehei lipase (4TGL) [119] showing the residues in the binding cleft, which is supposed to be in near contact with the substrate acyl chain. The inhibitor diethyl phosphate (DEP) is coloured red, and the lid residues are coloured green.

acetyl moiety in lipases is an elongated binding pocket (see Fig. 8, see also Fig. 6 for comparison of residues within the homologous lipases). Several residues pointing into the acyl-binding pocket have been mutated. Joerger et al. [54] introduced mutations in positions V206 and V209 along the acylbinding pocket, as well as the F95 in the end of the binding pocket and the F112 in the bottom of the binding cleft. The double mutation V206/V209 increased the activity three times against tricaprylin relative to triolein. F95D increases the relative activity towards tricaprylin, whereas the variant F112W lowers the activity. The V209T lowers the activity on triolein and the V209W increases the relative activity for tricaprylin and tributyrin two and four times, respectively. Further, Klein et al. [67] introduced a salt bridge in the acyl-binding cleft by making the double variant F95D/F214R. An introduction of a hydrophilic side chain as Gln in position 112 largely eliminated the activity. The double variant V206T/ F95D in the RDL prolipase gave an additive eect on substrate selectivity against single homoacylglycerols [66]. The pH prole of the double variant V206T/F95D on the other hand is changed compared to the wild type, whereas none of the single mutants showed the change in pH prole. A variant in position 85 of Fusarium solani pisi cutinase showed shifts in chain length specicity compared to the wild-type enzyme [69,70]. The A85F was chosen due the positioning at the entrance to the active site. The specic activity increased for chain length longer than C7, but decreased for chain length shorter than C7, on a substrate analogue with an ester with varying chain length in position sn1, an acyl amide in sn2 and acyl group in position sn1, the last two non-degradable for the cutinase. The chain length specicity was also addressed in Pseudomonas mendocina cutinase [57]. An increase in ratio of paranitrophenyl-butyrate/paranitrophenyl-caprylate was obtained by the substitution S205A and F207 to A or H, and a decrease in the ratio by S205T and Q127 to L, R or T. Geotricum candidum produces two isoenzymes that are 86% identical, of which GCL1 has preference for long chained and GCL2 for medium length chained unsaturated acid lipids [71]. Substitutions of a segment between residues 349 and 406 in GCL1 by the homologous part of GCL2 transferred the GCL1 into a GCL2 type activity, whereas the opposite seg-

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ment transfer did not fully result in GCL1 activity in GCL2. By making site-specic mutations in GCL1 in that region in the entrance to the active site, L358F and L357A/L358F lowered the long chained (triolein)/short chain (trioctanoin) activity ratio. 3.4. Positional specicity and enantioselectivity Scheib et al. [72] was able to predict by computer modelling and docking, the selectivity for sn1 or sn3 position in triacylglycerol analogues for two variants in positions L258 and L254 in Rhizopus oryzea lipase. By increasing the binding site with L258A and L258S, the sn1 hydrolysis was favoured. By decreasing the binding site (L258F and L258F/L254F) the sn3 position was favoured. The positional selectivity was shown by Rogalska et al. [4] to be dependent on the substrate type in lipid monolayers, and thus also to the lipid presentation. This issue has not been addressed by protein engineering as yet. Enantioselectivity has been discussed for mutations in position 87 and 89 in the lid of Humicola lanuginosa lipase [59]. These positions in the lid were shown to be important for the enantioselectivity. Scheib et al. has studied the resolution of diradylglycerols [73] and triradylglycerols [72] in Rhizopus lipase. Recently, work on developing an increased specificity for one of the enantiomers was done using error-prone PCR mutagenesis in Pseudomonas uorescens esterase [74] and in Pseudomonas aeruginosa lipase [75,76]. For the Pseudomonas aeruginosa lipase the libraries were screened in special developed assays. A dramatic increase in enantioselectivity was achieved, showing an ee% increase from 2% to 81% within four mutant generations. 4. Variants addressing stability 4.1. Thermostability Proline residues exhibit special dihedral angles for the P and i angles in the polypeptide backbone. The cyclic proline residue lowers the entropy of unfolding and thus stabilises the protein. Proline substitutions have been made in Humicola lanuginosa lipase in positions where the P and i dihedral angles are ap-

propriate for a Pro residue [77]. The temperature stability has been increased by 2C for the mutation G225P in Humicola lanuginosa lipase measured by dierential scanning calorimetry [77]. Increased temperature stability of 12C has been obtained in the Penicillium camembertii lipase [78]. A cystine bridge was introduced by homology to Humicola lanuginosa lipase, which has a cystine bridge spanning from the N-terminal part to the C-terminal part of the sequence, between residues C22^C268 (see Fig. 3 for sequence comparison). The optimum temperature for activity was increased by 10C and the pH optimum was changed to a more acidic pH by 0.7 units. In Fusarium heterosporum lipase, a lipase homologous to Rhizomucor miehei lipase, a Cterminal extension was shown to increase temperature stability. A level of approximately 100% residual activities after incubation at elevated temperatures are shifted about 10C from approximately 70C to 80C [79]. Even after cleavage of the extension, the non-covalently bound peptide improved the temperature stability of the lipase. The Candida antarctica B lipase showed a thermal stability increase of 4C by introduction of the variant T103G in the consensus sequence GXSXT [49]. 4.2. Protease stability The use of lipase in detergent has prompted the interest in adding the lipase together with proteases. The proteolytic stability of the lipase can be improved by protein engineering. Two enzymes have been improved by protein engineering: Pseudomonas glumae lipase [80] and Humicola lanuginosa lipase [81]. The concept was in one case to add proline residues next to the scissile peptide bond in the lipase. In Humicola lanuginosa lipase proline substitutions were made in position 212, which was found to be PP2 position after cleavage with the subtilin protease, Savinase. In another concept protease non-degradable loops were `transferred' to the susceptible lipase loop site. A loop consisting of the residues NGYD was exchanged for a loop containing the residues GASG in Humicola lanuginosa lipase [81]. In both cases the protease sensitivity was decreased. Proline substitutions were made in positions 154 and 150 in Pseudomonas glumae lipase. Also arginine residues were introduced into the PP1 position, as deg-

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radations of test peptides indicated a lower hydrolysis rate for arginine in position PP1 to the cleavage site. In the Pseudomonas glumae lipase the scissile sites were in the hinges of the lid region, and thus, the mutant showed beside the protease stability, also changes of the functionality of the enzyme. Often after the primary cleavage site in the lipase sequence is identied, and a substitution of a proline into the scissile site has been made, the resulting protein will have a new protease susceptible site. By combination of variants the protease stability can be improved suciently, but not always with the lipase functionality and specicity intact. 4.3. Oxidative stability Oxidative stability highly addressed for protease and amylase, has not been of major interest for the lipases, since many lipases are rather stable in oxidative reagents. Exchange of methionines to other residues in Pseudomonas sp. has been made in order to modify the inactivation of the lipase by oxidation in oxidative detergents [82,121]. The variant M72L in the Candida antarctica B lipase showed an increased stability towards oxidation by peroxyoctanoic acid, but also showed an activity lowered to half of wild-type enzyme in synthesis of octyl dodecanoate [49]. 5. Mutations addressing lid function The lid(s) or ap(s) of the lipases has always attracted a big interest. Deletions or substitutions in the lid have been attempted in many lipases including Human lipoprotein lipase, Human pancreatic lipase and Humicola lanuginosa lipase, with the purpose of changing the lipase function or changing the conformation of the lid or ap. Covalently trapping of the lid in an open conformation has been attempted resulting a partly open lid, but also a decrease in activity [83]. Transfer of the ap between Human pancreatic lipase/guinea pig pancreatic lipase-related protein 2 restores phospholipase activity [84]. The removal of the lid in the Humicla lanuginosa lipase resulted in an inactive lipase [83], whereas deletion of the ap in Human pancreatic lipase resulted in active lipase [85]. This dierence is probably reected by

the dierence in loop placement. For Humicola lanuginosa lipase the loop is covering a long stretch between scaold contact, whereas in the human pancreatic lipase the scaold connections for the ap is in close contact. A partly deleted ap in the Human pancreatic lipase from residue 248^257 was also fully active and showed a slightly increased binding to mixed micelles of tributyrin and taurocholate. A deletion of residues 240^260 spanning the entire ap, resulted in a lipase having a lag phase on triolein substrate and not on tributyrin. The wild-type enzyme has no lag phase. This phenomenon was probably due to improper interaction with the colipase. The issue was also addressed by the variants R256G/ D257G/Y267F/K268E [86], which did not abolish the interaction with colipase, but rather the ap domain conformation abolishing the proper interaction with the lipid. Making chimaeric enzymes by lid exchanging of Human hepatic and lipoprotein lipase demonstrated dierences between the relative specic activities of the enzyme [87]. Domain exchange or chimers has been made between human pancreatic lipase and Human lipoprotein lipase [88] and between guinea pig pancreatic lipase related protein 2 (GPLRP2), which has a much reduced size of ap, and the Human pancreatic lipase [89]. Mutations in the lid have been made to address the charge system of the lid and the remainder of the lipid contact zone have been made in Humicola lanuginosa lipase. By substitution of the E87 to Ala the importance of the charged glutamate was analysed [59]. This was based on the homology to the charge system in most other homologous lipases as Rhizomucor miehei lipase, which has an arginine is this position, and which also was found to be inhibited by guanidium, resembling the guanidium of the arginine side chain and chemical modication of the lid arginine [90]. Further analysis by computer simulations indicated the importance of the interaction of charges in the opening of the lid [91,92]. Variants have been made in Staphylococcus hyicus based on a sequence alignment between Staphylococcus lipase and Pseudomonas lipase indicating a putative lid region to be present between residue 323 and 333 [93], and the kinetic data of the mutations L326F and L326A is suggested to support that hypothesis.

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The functionality of the lid has been addressed using variants with only one tryptophan present in the lipase and placed centrally in the lid [94]. By uorescence analysis the environment of the lid and the mobility has been addressed by addition of surfactants to the lipase. Fluorescence studies have also been made of the variant W89F [95]. 6. Mutations addressing macroscopic substrate specicity Measuring lipase activity for the macroscopic substrate is an issue to address as well as the monosubstrate type. The surface of lipid has importance for the activation of the enzyme (see above) and for the presentation of the substrate to the enzyme. Also the surface pressure is important for the enzyme stability as well as its specicity [4]. In order to analyse binding of the lipase to the real lipid substrate, an inactive variant with only one Trp in the lid has been made. In Humicola lanuginosa lipase the variant S146A [96^98] has been made addressing the interaction with the macroscopic substrate or the interaction with lipid surface. The binding has been the main concern in vesicular systems. Further the Trp. The multivariant W221H,/W261H/W117F and the single variant W89F are being used as reporters for the substrate binding or rather the lipase binding to the substrate surface [94,99]. In PLA2 cysteine variants have been made to cover most the PLA2 surface. The cysteines have then been derivatised with spin-labelled groups. ESR measured the quenching of the spin signal upon binding of the PLA2 to the lipid surface. In this way the lipid contact zone of the PLA2 was suggested [38]. The variant V3W in Human (Group Iia) PLA2 was shown to have 250 times increase in activity against unilamellar vesicles of phosphatidylcholine vesicles [100]. It was suggested that the Trp made the enzyme more able to penetrate the phosphatidylcholine lipid. Interestingly the activity was lowered against anionic substrates. Experiments using attenuated total reection Fourier transformed infrared (ATR-FTIR) and oil-drop tensiometry indicated that the interfacial binding rather than the specic activity of Fusarium solani pisi cutinase variants is the determining factor of the interfacial tension change [29].

7. Mutations addressing calcium binding The lipases from Pseudomonas species has a calcium-binding site, and by homology the Staphylococcus species was suggested to have a calcium site. Staphylococcus hyicus lipase has been made calcium independent by mutations in position 357 and 354 [101]. The mutations were made based on sequence alignment to other lipase sequences, including the Pseudomonas glumae lipase sequence, of which an X-ray structure is present showing a calcium-binding site, and further experimental data on the Staphylococcus lipase suggested a calcium binding. The D357K remained active in absence of calcium ions. Isothermal titration calorimetry experiments indicated that the independence of calcium was coupled to a disappearance of calcium binding. The importance of calcium for activity of the Pseudomonas cepacia lipase has been probed by making mutations in positions D242 and D288 thus resulting in a less active enzyme [102]. 8. Mutations addressing detergent use and surfactant compatibility In detergents, anionic and non-ionic surfactants are the main components. The anionic compatibility of Fusarium solani pisi cutinase has been addressed by mutations in position 172, 17 and 196 based on surface charge analysis [70]. Substitution resulting in a more positively charged surface, N172K, was found to be less stable than the wild-type cutinase. Introducing negative charged residues in positions 17 and 196 (R17E and R196E), resulted in an enzyme with increased stability against the anionic surfactant lithium dodecyl sulphate. Also calcium chelators are added in high amounts to detergents. These chelators remove most of the free calcium ions in the wash liquor. Lipases from Pseudomonas species have a structurally strong calcium-binding site, whereas a lipase like Humicola lanuginosa lipase contains no calcium. However, the activity of Humicola lanuginosa lipase does depend on calcium presence, especially when degrading long chained fatty acids glycerols. Surface engineering of the Humicola lanuginosa lipase has been made in order to overcome the dierent important issues for the lipase in detergents

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[81,103]. The stability and compatibility in surfactant mixtures, the low calcium concentration present in detergent, as well as the standard lipase functionality on a multi-substrate surface has been addressed (see above). The Humicola lanuginosa variant D96L is less dependent of calcium presence and has an increased stability in the presence of non-ionic and anionic surfactants. Many variants have been made, that function in various detergents and which are independent of calcium for activity [104] For the Pseudomonas glumae lipase performance and stability has also been addressed [105]. Site-directed mutagenesis and random mutagenesis has been used to make variants in the Pseudomonas mendocina cutinase [106]. Libraries of 14 000 in size were made using poisoning by synthetic primers in a specic region. A primary assay on plates and a secondary assay for hydrolysis rate on cloth immobilised radioactive triolein was used for evaluation. The variants R202L/ Y203L showed increased activity, and the position Y203 was shown substituted to ten other variants with the V as the best residue in the secondary hydrolysis assay. 8.1. Variants, X-ray structures and dynamics Several cysteine variants of the cutinase Fusarium solani pisi have been made in order to obtain heavy atom derivatives for X-ray structure elucidation [107,108]. With a high number of variant structures a deeper understanding of the Fusarium solani pisi cutinase structure-activity relations ship has been established [108]. The superimposition of all the structures revealed a dynamic behaviour of the cutinase. The double variant L81G/L182G and the variant L81W have been analysed thoroughly by surface characterisation using computer programs [109]. The lower activity shown by the L81W variant was explained by a partly blocking of substrate entering the active site. Whereas the L81G/L182G increased the access to the active site, it was found to have decreased hydrophobicity and interestingly the B-factors of the variant in the loop 180^188 was decreased. This nding is surprising as the smaller glycine residues were expected to increase the mobility of the loop. Variants of Pseudomonas mendocina cutinase have been crystallised under dierent conditions as pH, buers and precipitants, resulting in

dierent crystal forms. Small but signicant dierences were found in the crystal structures, many of these being due to lattice interactions [106]. 8.2. Methods of mutation strategy used Although site-directed mutagenesis has been widely used and is extremely helpful in analysing lipase function [70,81,103], the use of more random or semi random approaches are increasingly seen for many proteins as well as for lipases [74,76,81,104, 106,110^112]. The interest in industry in highthroughput screening using robotics has also been found attractive in the scientic community to demonstrate the possibilities to improve and further develop characteristics not hitherto found simple or feasible to do by site-directed techniques. Further, the diculty in prediction of protein characteristics has increased the hope to the use of these more random techniques. The many possibilities in total random mutations, and the directionality within the socalled random techniques as error-prone PCR, has prompted to develop localised random mutagenesis [81,104]. This technique combines the structural knowledge with the random methodology to optimise the DNA libraries, and to avoid pre-screening and selection or screening of inactive variants. The random type techniques have shown good results, and will be helpful in further understanding the structural aspects, which are often dicult to predict [111]. An important part for using the directed evolution or localised random mutagenesis is the screening assays [76,106,110,113,120]. The reported assays are often directed against stability or function under conditions, where little or no eect is seen for the parent enzyme. Future development within assays will be a very important part for further development of any enzyme. For lipases and other enzymes, with the special characteristics of surface associated activities, assay development demands a special attention, in order to obtain the wanted characteristics of the enzyme. Acknowledgements Thanks to Shamkant Anant Patkar, Andreas Larvig Andersen, Sanne Ormholt Schrder Glad and

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