Further inhibition studies on guanidinobenzoatase, a trypsin-like enzyme associated with tumour cells

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasminogen activator, plasmin, thrombin and a newly described tumour associated enzyme specific for guanidino phenylalanine residues. These conclusions have been derived from inhibition studies employing 4-methyl-p-guanidinobenzoate as substrate. Three active site titrants for trypsin have been shown to be good substrates for guanidinobenzoatase. A new active site titrant for trypsin, rhodamine bisguanidinobenzoate, can also be used to assay guanidinobenzoatase in a stoichiometric manner. This active site titrant can be employed to label guanidinobenzoate on the surface of leukaemia cells.     

The active site titration of proteinases by using alpha 2-macroglobulin and high-performance liquid chromatography.

The active site titration for various proteinases relies on the development of optimal enzyme titrants for each proteinase, but these titrants are only available for a limited number of proteinases. We have described a new active site titration method applicable to various kinds of endoproteinases using small quantities of the enzymes. This method was carried out by using alpha 2-macroglobulin (alpha 2M) as a titrant and a high-performance liquid chromatography (HPLC) system. When the proteinase solution was treated with alpha 2M, the active proteinase was trapped by alpha 2M. In this reaction alpha 2M does not usually complex with inactive proteinase. After the reaction of proteinase with an excess of alpha 2M, the reaction mixture is applied to an HPLC gel column to separate the uncomplexed enzyme from the one complexed with alpha 2M. The active proteinase is complexed and eluted with alpha 2M, but the inactive proteinase is eluted at the original elution volume. The same amount of the enzyme was also applied to the column. From the decrease of the peak height at the elution position of the uncomplexed proteinase, we can estimate the ratio between enzymatically active proteinases and total proteinases. To test the usefulness of this method, we applied this method to chymotrypsin and trypsin whose activities were predetermined by conventional active site titration, and there was good agreement between both results. With this new method, we can estimate a proteinase activity with as little as 200 ng of the enzyme, a very small amount compared with those required in conventional methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the catalytic properties of thrombin and trypsin by kinetic analysis on the basis of active enzyme concentration.

The interaction of bovine thrombin [EC 3.4.21.5] with synthetic substrates and products was studied. The enzyme was purified from Parke-Davis topical thrombin. The purification process afforded some preparations with different clottin specific activities but with similar esterase specific activities. The preparation having highest clotting specific activity and that having lowest clotting activity were tentatively named thrombin-C and thrombin-E, respectively. Kinetic parameters for the hydrolysis of synthetic substrates and normality titrants were determined on the basis of active enzyme quantity, which was assayed by means of a fluorometric normality titrant. It was shown that thrombin-E was acylated by the substrates more slowly than thrombin-C, while deacylation proceeded at similar rates in the two preparations. The results were also compared with those obtained with bovine trypsin [EC 3.4.21.4]. The acylation rates of both thrombin preparations were markedly lower than that of trypsin, while the deacylation rates of the former were only slightly lower than that of the latter. The effects of various product-type inhibitors, such as benzyloxycarbonyl-, benzoyl-, and tosyl-L-arginine, were also examined. Thrombin was affected by these inhibitors not competitively, though trypsin was inhibited competitively.     

Characterization of the mouse sperm plasma membrane zona-binding site sensitive to trypsin inhibitors

The first contact of mammalian gametes is the binding of the spermatozoon to the zona pellucida of the egg. Previous work has shown that binding of the spermatozoon to the zona in the mouse occurs prior to the acrosome reaction and that trypsin inhibitors block this initial binding. This suggests that the sperm surface contains a trypsinlike binding site that functions by an active site mechanism to effect initial zona binding. When suspensions of twice-washed spermatozoa were incubated with the serine protease active site titrant, 4-methylumbelliferyl p-guanidinobenzoate (MUGB), the titrant was hydrolyzed at a rate of 8 pmoles/min-10(6) cells. MUGB was found to inhibit the binding of spermatozoa to the zona pellucida. The degree of inhibition and the rate of hydrolysis of MUGB by washed spermatozoa depend on the concentration of titrant, with half maximal effects at 13 microM and a linear correlation with r = 0.99. The analogous lysyl and arginyl trypsin substrates containing 7-amino-4-methylcoumarin as the fluorogenic leaving group were not hydrolyzed under the same conditions and did not inhibit zona binding. Both binding of sperm to zona-intact eggs and the hydrolysis of MUGB by sperm are inhibited by p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, and acid-solubilized zonae. The linear correlation coefficients of the inhibition of sperm binding and MUGB hydrolysis by these three substances are greater than 0.92. This "trypsinlike" sperm site is essential for sperm binding to the zona: its stereospecificity is unique in that it reacts with trypsin inhibitors but not with trypsin substrates.     

Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration

Several esters of 4-methylumbelliferone and 2-naphthol were synthesized and examined as possible spectrofluorimetric titrants for bovine alpha-chymotrypsin, trypsin, thrombin, Factor Xa and for subtilisin Novo. 4-Methylumbelliferyl p-guanidinobenzoate hydrochloride (MUGB) is a satisfactory titrant for alpha- and beta-trypsin, thrombin and Factor Xa and 4-methylumbelliferyl p-(NNN-trimethylammonium)cinnamate (MUTMAC) is a good titrant for alpha-chymotrypsin. The amount of enzyme used for spectrofluorimetric titration is 0.02-3.00nmol and the amount of 4-methylumbelliferone liberated is independent of the concentration of titrant and stoicheiometrically equal to the amount of enzyme used. Results obtained with MUGB and MUTMAC have been checked by spectrophotometric titration with p'-nitrophenyl p-guanidinobenzoate hydrochloride and p-nitrophenyl N(2)-acetyl-N(1)-benzylcarbazate respectively. p-Nitrophenyl N(2)-acetyl-N(1)-(9-anthrylmethyl)carbazate has been synthesized; it did not react with alpha-chymotrypsin. A satisfactory spectrofluorimetric titrant for subtilisin Novo was not discovered.     

N alpha-dansyl-L-arginine 4-phenylpiperidine amide. A potent and selective inhibitor of horse serum cholinesterase

The relationship between chemical modifications of arginine derivatives and inhibitory activity to horse serum cholinesterase (BuChE) was investigated. It provided a new insight into the topography of the active site of BuChE. 1) BuChE has the hydrophobic binding pocket, the depth of which corresponds to the length of ethylpiperidine. 2) In the opposite side to the hydrophobic binding pocket, BuChE has a certain entity which repulses carboxyl group at the 2-position of piperidine of L-arginine piperidine amide. 3) The P site of BuChE can allow 4-propyl and 4-phenyl group attached to piperidine. Comparison of the results with those of thrombin and trypsin clearly revealed similarities and dissimilarities among BuChE, trypsin, and thrombin in the active site topography, and hence, we introduce a new selective inhibitor for BuChE, N alpha-dansyl-L-arginine 4-phenylpiperidine amide. It inhibits BuChE strongly (Ki = 0.016 microM), whereas it inhibits trypsin, thrombin, plasmin, and glandular kallikrein only weakly and shows actually no inhibition on acetylcholinesterase from the human erythrocyte. In addition, the new inhibitor becomes highly fluorescent when bound with BuChE, indicating that the compound is an ideal probe of the interactions of BuChE as well as a titrant of it.     

A fluorogenic active-site titrant for serine proteases

The synthesis of 3'-(4-guanidinobenzoyloxy)-spiro(isobenzofuran-1(3H),9H-[9H]xan then]-3-one or 3HFGB, an active-site titrant for serine proteases, is described. 3HFGB has two advantages over previously described titrants. It is very sensitive (it can measure serine proteases at the 10(-11)-10(-12) M level), and it can be used at stoichiometric ratios of titrant to protease of unity or less. Moreover, the fluorogenic leaving group, 3'-hydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3- one, is suitable for use in a range of esteropeptidase substrates. The use of 3HFGB in the active-site titration of trypsin is described.     

The preparation of fully active chymopapain free of contaminating proteinases

Chymopapain (EC 3.4.22.6) was purified from commercially available dried latex of papaya (Carica papaya) by extraction at acidic pH, cation-exchange chromatography and active site-directed affinity chromatography on immobilized alanyl-phenyl-alaninaldehyde semicarbazone, with elution by mercuric chloride. The product was found by immunoassay to be essentially free of the other cysteine proteinases from papaya, including papaya proteinase IV, and was fully active. The rate of alkylation of the active site cysteine of chymopapain by iodoacetate was found to be sufficiently rapid and selective for this reagent to be used as an active-site titrant.     

Arginine substitution by alanine at the P1 position increases the selectivity of CmPI-II,a non-classical Kazal inhibitor

CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine.     

(p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases

p-(Amidinophenyl)methanesulfonyl fluoride (p-APMSF) has been synthesized and shown to be a specific, irreversible inhibitor of the class of plasma serine proteases which demonstrate substrate specificity for the positively charged side chains of the amino acid lysine or arginine. In equimolar concentration, this compound causes immediate and complete irreversible inhibition of bovine trypsin and human thrombin. A 5-10-fold molar excess of reagent over enzyme is required to achieve complete irreversible inhibition of bovine Factor Xa, human plasmin, human C1-r, and human C1-s. the Ki of p-APMSF for all of the above-mentioned proteases is between 1 and 2 microM. In contrast, p-APMSF in large molar excess does not inactivate chymotrypsin or acetylcholinesterase. The unique reactivity of p-APMSF has been further shown in comparison with the related compound p-nitrophenyl (p-amidinophenyl)methanesulfonate which is an active-site titrant for thrombin but reacts poorly with Factor Xa, C1-r, and C1-s and is not hydrolyzed by bovine trypsin or human plasmin. Similarly, (p-amidinophenyl)methanesulfonate has a Ki of 30 microM for thrombin but is a poor inhibitor of trypsin, Factor Xa, C1-r, C1-s, and plasmin. Studies with bovine trypsin have demonstrated that the inhibitory activity of p-APMSF is the result of its interaction with the diisopropyl fluorophosphate reactive site. The unique reactivity of this inhibitor classifies it as one of the most effective active site directed reagents for this class of serine proteases. Collectively, these results suggest that the primary substrate binding site of these enzymes, which share a high degree of structural homology, do in fact significantly differ from each other in their ability to interact with low molecular weight inhibitors and synthetic substrates.     

Thioltrypsin. Chemical transformation of the active-site serine residue of Streptomyces griseus trypsin to a cysteine residue.

The active-site serine residue of Streptomyces griseus trypsin was converted to a cysteine residue, and the product, thioltrypsin, was purified through two chromatographic steps with organomercurial-Sepharose and soybean trypsin inhibitor-Sepharose as specific adsorbents. The purified preparation of thioltrypsin was found to contain a single residue of cysteine and to react with almost equimolar amounts of normality titrants. It exhibited only traces of catalytic activity toward typical trypsin substrates such as Nalpha-tosyl-L-arginine methyl ester, whereas it retained some activity toward "active ester" substrates such as Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester. The activity was inhibited by sulfhydryl-blocking reagents, but no inhibition was observed by reagents reactive with the active hydroxyl group of serine proteases. Leupeptin, a natural trypsin inhibitor of peptidyl nature, also inhibited thioltrypsin. Some difference in the mode of leupeptin inhibition, however, was detected between trypsin and thioltrypsin. The bindings of small synthetic ligands and soybean trypsin inhibitor to thioltrypsin were compared with those to trypsin.     

The reversible thiol-disulphide exchange of trypsin and chymotrypsinogen with a tumour-derived inhibitor. Kinetic data obtained with fluorescein-labelled polymeric collagen fibrils and casein as substrates.

Ehrlich ascites cells contain a cytoplasmic inhibitor of both trypsin and the granule neutral protease and possess a reactive thiol which interacts with an important disulphide bond in trypsin, resulting in the formation of the trypsin-inhibitor complex. When a fixed quantity of trypsin was completely inhibited by addition of the cytoplasmic inhibitor, the trypsin could be re-activated by the addition of either trasylol-trypsin or chymotrypsinogen. Since trasyloltrypsin, chymotrypsinogen (and any derived chymotrypsin) has no ability to solubilise fluorescein-labelled peptides from the substrate, the appearance of trypsin activity was probably due to a non-enzymic exchange reaction, in which these inactive forms displaced trypsin from the trypsin-inhibitor complex. Kinetic data suggest that this displacement was a time-dependent equilibrium reaction controlled by the relative concentration of the reacting species.     

An inexpensive device for sample stirring in VSU Zeiss spectrophotometers. Its use for kinetic measurements and active site titrations with immobilized enzymes.

The construction of a simple and effective sample stirring device for commercial spectrophotometers and its use for continuous kinetic measurements and active site titrations with immobilized enzymes is described. Sepharose-bound leucine aminopeptidase and trypsin were selected as model enzymes to test the performance of the magnetic stirring equipment. Kinetic parameters of insolubilized leucine aminopeptidase using L-leucine p-nitroanilide as substrate and the catalytic site concentration of matris-bound trypsin using p-nitrophenyl p'-guanidinobenzoate as active site titrant could be evaluated without significant interference from the turbidity of the stirred Sepharose suspension. The problem of grinding of the support material could be overcome. Both unbound native and carrier-fixed enzyme may be reacted under identical conditions with similar convenience and sensitivity.     

用定位突变探讨胰蛋白酶的稳定性

应用定位突变的方法,对鼠胰蛋白酶分子中易自溶位点的氨基酸残基进行改造,以探索提高胰蛋白酶稳定性的可能。将鼠胰蛋白酶原cDNA从质粒pTRAP上切下,插入载体M13mp8,在E.coli JM 101菌株中转化、复制后用以转染RZ1032缺陷型菌株,利用含U模板,进行按实验目的设计寡聚核苷酸引物引导的定位突变,将易自溶位点Ary105改造为Leu或Gly。经酶切和序列测定,证明在设计位点发生了预期的碱基突变。为了检查活性方便,又将含突变型胰蛋白酶cDNA从pTRAP上切下,插入另一个表达载体pTN中,转化入E.coli SM 138宿主中进行表达,表达产物分泌到围膜间隙,作专一性底物TAME活性胶方法检测,证明改造后的表达产物具有胰蛋白酶活性。对突变与野生型胰蛋白酶进行了初步比较。     

Exploring the protease mediated conformational stability in a trypsin inhibitor from Archidendron ellipticum seeds

A Kunitz proteinase inhibitor from Archidendron ellipticum seeds (AeTI) was purified and complexed with bovine trypsin and chymotrypsin. The stoichiometric stability of AeTI with its interacting proteinases was then investigated using spectrophotometric, size exclusion chromatography (HPLC system), Western blotting and circular dichroism (CD) studies. All the methods were remarkably similar in revealing the preference of trypsin over chymotrypsin by AeTI for complex formation. Both Western blotting as well as spectrophotometry based assays for competition experiments indicated that trypsin displaces chymotrypsin from a previously formed AeTI-chymotrypsin complex. Chemical modification of lysine and arginine by TNBS and CHD treatments, respectively, suggested a lysine as the active site residue and also indicated the presence of a single protease-binding site for AeTI. CD of native AeTI showed a sharp minimum at 200 nm and deconvolution of the CD spectra revealed it to be an unordered protein possessing high beta-sheet content. Complex formation of AeTI with trypsin induces a fractional switchover of its unordered structure towards the beta-sheet fraction but lacked any such conversion in the presence of chymotrypsin. Prolonged exposure of excess trypsin generates conformational modifications both in the secondary and the tertiary structures.     

Crystal structure of cancer chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 A resolution. Structural basis of Janus-faced serine protease inhibitor specificity

Understanding molecular recognition on a structural basis is an objective with broad academic and applied significance. In the complexes of serine proteases and their proteinaceous inhibitors, recognition is governed mainly by residue P1 in accord with primary serine protease specificity. The bifunctional soybean Bowman-Birk inhibitor (sBBI) should, therefore, interact at LysI16 (subdomain 1) with trypsin and at LeuI43 (subdomain 2) with chymotrypsin. In contrast with this prediction, a 2:1 assembly with trypsin was observed in solution and in the crystal structure of sBBI in complex with trypsin, determined at 2.3 A resolution by molecular replacement. Strikingly, P1LeuI43 of sBBI was fully embedded into the S(1) pocket of trypsin in contrast to primary specificity. The triple-stranded beta-hairpin unique to the BBI-family and the surface loops surrounding the active site of the enzyme formed a protein-protein-interface far extended beyond the primary contact region. Polar residues, hydrophilic bridges and weak hydrophobic contacts were predominant in subdomain 1, interacting specifically with trypsin. However, close hydrophobic contacts across the interface were characteristic of subdomain 2 reacting with both trypsin and chymotrypsin. A Met27Ile replacement shifted the ratio with trypsin to the predicted 1:1 ratio. Thus, the buried salt-bridge responsible for trypsin specificity was stabilised in a polar, and destabilized in a hydrophobic, environment. This may be used for adjusting the specificity of protease inhibitors for applications such as insecticides and cancer chemopreventive agents.     

Characterization and partial purification of the sodium-potassium-ATPase inhibitor released from cultured rat hypothalamic cells

An inhibitor of sodium-potassium-ATPase has been partially purified from the culture medium obtained from hypothalamic cells maintained in a capillary membrane perfusion system, and some of the properties of this inhibitory factor have been investigated. Gel filtration (Sephadex G-25 Superfine) of heat-treated medium (80 degrees C for 10 min) resulted in elution of inhibitory activity in the post-salt fraction. These fractions inhibited active (i.e. sodium-potassium-ATPase-mediated) sodium transport in intact human erythrocytes, displaced [3H]ouabain from its binding site, and directly inhibited canine kidney sodium-potassium-ATPase as measured by NADH oxidation. High-performance liquid chromatography (on Hypersil ODS) of these fractions after desalting yielded one region which showed inhibitory activity on all three assays. Inhibition of sodium-potassium-ATPase was dose-related and filtered through an Amicon UM10 membrane. Incubation of this material with dispase, carboxypeptidase A, chymotrypsin, and prolidase destroyed inhibitory activity, whereas trypsin and leucine aminopeptidase were ineffective. These studies show that hypothalamic neurones release a low molecular weight heat-stable peptide which inhibits active sodium transport, ouabain binding, and sodium-potassium-ATPase.     

Equilibrium of Bowman-Birk inhibitor association with trypsin and alpha-chymotrypsin.

Association constants, enthalpies, and stoichiometries of Bowman-Birk soybean inhibitor for trypsin and alpha-chymotrypsin were measured in the pH range 4-8 at 25 degrees, 0.01 M Ca2+. The results are quoted in terms of moles of protease active sites, from active site titration. Enthalpies were obtained from calorimetry. The inhibitor was modified by carboxyl group modification, and by tryptic and chymotryptic attack. Association thermodynamics and stoichiometries of the modified inhibitors with both proteases were also determined. There is one independent site for each protease on the inhibitor protein. Modification decreases association to some extent, but does not appear to change stoichiometry or protease binding site independency. In the pH 4 region the association enthalpies are endothermic, of the order 6 kcal/mol for both trypsin and chymotrypsin. With increasing pH, the enthalpies decrease and become exothermic at pH 8 for chymotrypsin. Positive entropies, 50 cal mol-1 deg-1, occur at pH 4-5. They decrease as pH increases, but are always positive in sign. The observed to accompany the overall reaction, such as H+ transfer steps. The enthalpies and entropies probably compensate over the pH range 4-8, with a characteristic temperature of 390 plus or minus 30 degrees K. Estimates were made of the macromolecular Coulomb charge products in inhibitor-protease interaction. These range from about +5 to -60, over pH range 4-8, depending on the protease. Although intermolecular Coulombic forces cannot be easily delineated at the specific side chain level, they may operate at the macromolecule level.     

Catalysis by serine proteases and their zymogens. A study of acyl intermediates by circular dichroism.

p-Nitrophenyl p'-guanidinobenzoate and methylumbelliferyl p'-guanidinobenzoate, which are active site titrants for trypsin, and p-nitrophenyl p'-dimethylsulfonioacetamidobenzoate and methylumbelliferyl p'-trimethylammoniocinnamate, which are active site titrants for chymotrypsin, are also hydrolyzed by the respective zymogens. Hydrolysis in each case proceeds via the formation of acyl-zymogens. The acylation rates for the zymogens are 10(3)-10(7) times slower than for the enzymes whereas the deacylation rates of acyl-enzymes and acyl-zymogens are comparable. These findings are consistent with the idea that the diminished catalytic activity of these zymogens is due primarily to their distorted substrate binding sites. The circular dichroic spectra of the acyl-enzymes show induced negative ellipticities in the region of absorption of the acyl group, due to binding of the group in an asymmetric environment. The circular dichroic spectra of the acyl-zymogens do not, but conversion of the acyl-zymogens to acyl-enzymes changes the circular dichroic spectra to those characteristic of the acyl-enzymes. alpha-Carbamyl-epsilon-guanidinated trypsin is a derivative which resembles trypsinogen in lacking activity toward specific ester substrates but possessing low activity toward p-nitrophenyl p'-guanidinobenzoate. The circular dichroic spectrum of the acyl-enzyme formed during hydrolysis of p-nitrophenyl p'-guanidinobenzoate by this derivative resembles that of guanidinobenzoyltrypsinogen, and not that of guanidinobenzoyltrypsin. These circular dichroism studies confirm that the same serine residue is involved in catalysis by both enzymes and zymogens. They demonstrate directly that the acylating group is in a different environment in each and indicate that this specific environment is a determinant in the catalytic activity of each. Thus the circular dichroic spectra of these acyl intermediates provide a sensitive probe of the subtle conformational changes which occur on zymogen activation. The results support the previous conclusion that the major feature of the activation of trypsinogen and chymotrypsinogen is the rearrangement of the substrate binding site and that the appearance of a new amino terminus causes this rearrangement.     

Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester

The fluorescent organophosphorus esters, diethyl 4-methylumbelliferyl phosphate (1), ethyl hexyl 4-methylumbelliferyl phosphate (2) and ethyl 4-methylumbelliferyl heptylphosphonate (3) have been synthesized and evaluated as a sensitive active-site titrant of lipase. The phosphorus esters 1, 2 and 3 inactivated the lipase from Pseudomonas aeruginosa (LPL-312) with a second-order rate constant for enzyme inactivation (k(on)) of 1.8, 32 and 5600 s(-1) M(-1), respectively. The long-chain phosphonate 3 turned out to be the most potent inactivator of the lipase to release a stoichiometric amount of highly fluorescent 4-methylumbelliferone (4MU) as a leaving group. By using the phosphate 3 as an active-site titrant, the low concentration (4.5 nM) of the active lipase was titrated successfully. The highly sensitive active-site titration with 3 enabled the direct determination of the concentration of the active lipase expressed in a microscale culture medium. Although the expression level differed significantly from one culture to another, the titrated concentration of the active lipase was proportional to the apparent activity for all the independent cultures. The molecular activity calculated for the expressed lipase was found to be the same as that of the purified lipase. The present active-site titration method is widely applicable to the biocatalytic engineering of lipases such as directed evolution, site-directed mutagenesis, chemical modification and immobilization.