Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: II. Effect of temperature on reaction kinetics and general considerations of stability and productivity

Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RrnL), isolated from commercial preparations of Lipolase and Lipozyme, respectively, were solubilized in AOT-stabilized water-in-oil (w/o) microemulsions in n-heptane and aspects of their hydrolysis and condensation activity examined. The temperature dependence of HIL hydrolysis activity in unbuffered R = 10 microemulsions matched very closely that for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. Apparent activation energies were measured as 13 +/- 2 and 15 +/- 2 kJ mol / respectively. Condensation activity, however, was essentially independent of temperature over the range 5 degrees to 37 degrees C. The stability of HIL over a 30-day period was very good at all pH levels (6.1, 7.2, 9.3) and R values studied (5, 7.5, 10, 20), except when high pHs and low R values were combined. The excellent stability was reflected by the linearity of the productivity profiles which facilitate system optimization. The temperature dependence of RmL hydrolysis activity toward pNPC(4) showed a maximum at 40 degrees C and an apparent E(act) = 20 +/- 2 kJ mol(-1) was calculated based on the linear region of the profile (5 degrees to 40 degrees C). RmL esterification activity showed only a slight dependence on temperature over the studied range (0 degrees to 40 degrees C) and an apparent E(act) = 5 +/- 1 kJ mol(-1) was measured for octyl decanoate synthesis. Both RmL and HIL, therefore, have potential for application in low temperature biotransformations in microemulsion-based media. The stability of RmL over a 30-day period was good in R = 7.5 and R = 10 microemulsions containing pH 6.1 buffer, and this was reflected in the linearity of their respective productivity profiles. RmL stability was markedly poorer at more alkaline pH, however, and proved to be sensitive to relatively small changes in the R value. (c) 1995 John Wiley & Sons, Inc.     

Enantioselective esterification of 2-arylpropionic acids and trans-2-phenyl-1-cyclohexanol: Comparison between immobilised lipases from Candida rugosa and Rhizomucor miehei

2-Phenyl propionic acid, ibuprofen and trans-2-phenyl-1-cyclohexanol were resolved using commercial Rhizomucor miehei lipase (Lipozyme IM20) and Candida rugosa lipase produced in our laboratory immobilised on EP100 polypropylene powder. Important differences were found on the enantioselectivity of both lipases in esterification reactions. Candida rugosa lipase was more enantioselective in the resolution of the tested substrates, especially with trans-2-phenyl-1-cyclohexanol, whereas the lipase from Rhizomucor miehei did not show catalytic activity with this substrate. © Rapid Science Ltd. 1998     

Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa.

The substrate-binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme. Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme-substrate transition-state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.     

Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa

A kinetic study on esterification between d-glucose and l-phenylalanine catalysed by lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) in organic media investigated in detail showed that both the lipases followed a Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical double reciprocal plots and computer simulation studies showed that competitive double substrate inhibition took place at higher concentrations leading to dead-end inhibition in the case of RML and in the case of CRL, inhibition only by d-glucose at higher concentrations leading to dead-end lipase–d-glucose complexes. An attempt to obtain the best fit of these kinetic models through curve-fitting yielded in good approximation, the apparent values of important kinetic parameters, RML: k _cat = 2.24 ± 0.23 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 95.6 ± 9.7 mM, K _{m d-glucose} = 80.0 ± 8.5 mM, K _{i l-phenylalanine} = 90.0 ± 9.2 mM, K _{i d-glucose} = 13.6 ± 1.42 mM; CRL: k _cat = 0.51 ± 0.06 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 10.0 ± 0.98 mM, K _{m d-glucose} = 6.0 ± 0.64 mM, K _{i d-glucose} = 8.5 ± 0.81 mM.     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

The effect of ethanol on the kinetics of lipase-mediated enantioselective esterification of 4-methyloctanoic acid and the hydrolysis of its ethyl ester.

The Novozym 435(R) catalyzed esterification and hydrolysis reactions of 4-methyloctanoic acid (ethyl ester) were investigated. In both the hydrolysis and esterification reactions, the increase of ethanol concentration led to an increase in enantiomeric ratio (E). For hydrolysis of the ethyl ester, the E-value increased from 5.5 [0% (v/v) EtOH] up to 12 [20% (v/v) EtOH]. In case of esterification, the E-value was already 16 [14% (v/v) EtOH] and rose to 57 [73% (v/v) EtOH]. When combining these results of esterification and hydrolysis, an enantiomeric ratio of 350 can be estimated for the sequential kinetic resolution of 4-methyloctanoic acid. In this way, enantiopure 4-methyloctanoic acid could be obtained after two consecutive reaction steps.     

Enzymatic esterification reaction in organic media with continuous water stripping: effect of water content on reactor performance and enzyme agglomeration

A water removal system with in situ monitoring of the reaction medium turbidity was used to investigate the effect of water content on reactor performance and enzyme agglomeration. The performance of the system was tested by carrying out the lipase-catalyzed esterification of caprylic acid and n-butanol in cyclohexane. Enzyme agglomeration strongly depended on the water content in the solvent medium. By preventing agglomeration, the reactor could be operated in a continuous mode for 150 h without any loss of enzyme reactivity, whereas insufficient water stripping resulted in a gradual decrease of the conversion due to agglomeration of enzyme.     

毛豹皮樟叶水提物的活性成分及其清除亚硝酸钠和阻断亚硝胺合成的作用

亚硝酸盐在食材、食品加工和食品储存过程中普遍存在,它是亚硝基化合物合成的前体物质,在一定条件下亚硝酸盐极易转化为亚硝胺,二者均可致癌[1-2],因此,清除亚硝酸盐和阻断亚硝胺合成对于预防和控制癌症的发生具有重要意义。关于清除亚硝酸盐和阻断亚硝胺合成的研究已有较多报道,一些植物含有的天然活性成分,如植物中所含的多酚和黄酮类物质等,具有抗氧化、抗菌、清除亚硝酸盐和阻断亚硝胺合成的作用。顶羽菊也Acroptilon repens ( Linn.) DC.页[3]、黄花菜(Hemerocallis citrina Baroni)[4]和簕菜也Acanthopanax trifoliatus ( Linn.) Merr.页[5]等提取物具有清除亚硝酸盐和阻断亚硝胺合成的作用;黄俊生[6]从南姜也Alpinia galanga ( Linn.) Willd.页表皮中提取的色素也具有较强的清除亚硝酸钠和阻断亚硝胺合成活性。     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: I. Uniresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow fiber reactor

The kinetics of the hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow fiber reactor can be modeled in terms of a three‐parameter rate expression. This rate expression consists of the product of a two‐parameter rate expression for the hydrolysis reaction itself (which is of the general Michaelis–Menten form) and a first‐order rate expression for deactivation of the enzyme. Optimum operating conditions correspond to 30°C and buffer pH values of 7.0 during both immobilization of the enzyme and the hydrolysis reaction. Under these conditions, the total fatty acid concentration in the effluent oil stream for a fluid residence time of 4 h is approximately 1.6 M. This concentration corresponds to hydrolysis of approximately 50% of the glyceride bonds present in the feedstock corn oil. The fatty acid of primary interest in the effluent stream is linoleic acid. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 568–579, 1999.     

Effect of remained stem height on yield,quality of Inula helenium I. and on soil water content

To explore the reason causing low yield, poor quality of Inula helenium I., this paper investigated the influence of different remained stem heights on the yield, quality and water consumption of Inula helenium I. in Gannan plateau area using field single factor randomized block method. Research results showed that Inula helenium I. which was cut before blooming period in the last ten-day of July with remained stem height of 25?cm had the lowest water consumption, the best underground root traits (including main root length, root diameter, and root dry weight per plant), and the highest yield which was higher that control group by 18.73% (P?<?.01) Moreover, Inula helenium I. with remained stem height of 25?cm had the lowest ash content while the highest alantolactone content, therefore its quality was the best. The water use efficiency (WUE) of Inula helenium I. with remained stem height of 35?cm at September was the highest (1.12?kg?h?m^?2?mm^?1). However, in terms of biological yield WUE and economic yield WUE, Inula helenium I. with remained stem height of 15?cm was the highest. Therefore, it can be concluded that remained stem height from 15 to 25?cm is an ideal solution, which can not only save water, but also improve yield and quality of Inula helenium I.     

Reactivity of amino acids in the azo coupling reaction: I. Dependence of their reactivity on pH

Azo coupling reactions of N-α-acetylhistidine, N-α-acetyltyrosine, and N-α-acetyllysine with p-methylbenzenediazonium ion were investigated as model reactions to obtain information on the relative reactivity of the histidine, tyrosine, and lysine moieties of protein, separated from structural effects. The azo coupling yields of the amino acids increased as the pH of the reaction medium was increased, indicating that the ractive species are the imidazole anion of histidine, the phenolate anion of tyrosine, and the neutral ε-amino group of lysine. It was calculated, based on percentage yields of the azo products, that the imidazole anion is more reactive than the phenolate anion and the ε-amino group, respectively.     

Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: I. uniresponse kinetics

A calf pregastric esterase immobilized in a hollow-fiber reactor was employed to hydrolyze milkfat, thereby producing a lipolyzed butteroil. The reaction kinetics can be modeled by a two-parameter model of the general Michaelis-Menten form based on a ping-pong bi-bi mechanism; the rate of enzyme deactivation can be modeled as a first-order reaction. The initial concentration of accessible glyceride bonds, [G](O), was estimated by complete saponification of the substrate butteroil as 2400 mM. An extra sum of squares test indicated that not only the parameters of the kinetic generalized Michaelis-Menten model, but also the deactivation-rate constant varied significantly with pH. The optimum pH, for lypolysis is near 6.0 at a temperature of 40 degrees C because at this pH the rate of deactivation of the esterase is minimized.     

Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus beta-glucosidase: effects of donor concentration, water content, and temperature on activity and selectivity in hexanol.

The transglucosylation reaction catalyzed by wild-type beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-beta-glucoside to hexyl-beta-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The selectivity towards transglucosylation was quantified by the S value defined as follows: S = r(S) x a(W)/r(H) x a(hex) where r(S) and r(H) are the initial rates of transglucosylation and hydrolysis and a(w) and a(hex) are the thermodynamic activities of water and hexanol. The activity (rates of hydrolysis and transglucosylation) and the selectivity (S value) were measured as a function of pentyl-beta-glucoside concentration (5-240 mM), water content (1-100% v/v), and temperature (50-95 degrees C). All mutants had lower activity than the wild-type enzyme, but they had higher selectivity, which means that they provided a higher ratio of transglucosylation product to hydrolysis product. The largest increase in S-value (2.6 fold) was obtained by the F426Y mutant, which resulted in increased hexyl-beta-glucoside yield from 56% to 69%. In addition, the F426Y enzyme had higher selectivity over the wide range of temperatures tested. The activity of CelB wild-type and CelB F426Y increased as a function of water activity (a(w)), and complete activation by the water was obtained in a two-phase system with 20% water phase. In contrast to CelB wild-type, the F426Y mutant had transferase activity as low as a(w) = 0.29. Surprisingly, the S value increased with increasing water activity up to a(w) = 0.92. At still higher water content the S value decreased.     

Effect of subunit dissociation, denaturation, aggregation, coagulation, and decomposition on enzyme inactivation kinetics: I. First-order behaviour

Enzyme inactivation kinetics typically follows what would appear to be simple first-order behavior. However, the inactivation process is known to involve a number of reversible (decomposition, denaturation) as well as irreversible (decomposition, aggregation, and coagulation) reactions. These reactions can combine to form a wide variety of reaction pathways which can potentially demonstrate complex inactivation kinetics. However, it was shown that with appropriate assumptions with regard to the relative magnitudes of the various reaction rates, many complex inactivation pathways can demonstrate apparent first-order behavior. Thus, with this analysis, a more accurate interpretation of the slope of an activity versus time semi-log plot can be obtained. (c) 1992 John Wiley & Sons, Inc.     

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60 ± 5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved “burst” phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25 s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6 s) detected by all the spectroscopic probes, which occurred subsequent to an initial “burst” phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed. Protein 32:296–303, 1998. © 1998 Wiley-Liss, Inc.     

Effect of subunit dissociation, denaturation, aggregation, coagulation, and decomposition on enzyme inactivation kinetics: II. Biphasic and grace period behavior

A model previously developed to characterize enzymatic in activation behavior was used to explain the non-first-order biphasic and grace period phenomena that are often observed with oligomeric enzymes. Luciferase and urease were used as model enzyme such as luciferase, the oligomer initially dissociates reversibly into two native monomer species. These native monomers can then reversibly denature and irreversibly aggregate and coagulate. With the hexamer, urease, two trimers are formed that can subsequently aggregate to form an inactive hexamer. The dissociated monomer species of luciferase do not possess catalytic activity, so the inactivation mechanism, is biphasic; the first slope of a first-order kinetic plot is influenced by the reversible oligomer/monomer/denatured-monomer transition. Whereas the second slope is associated with either irreversible aggregation or coagulation. In contrast, the trimer of urease has the same activity as the hexamer; therefore, during the intitial hexamer-trimer transition, little activity loss occurs. However, as the trimer concentration increases, activity decreases as a result of trimer aggregation. As a result, grace period inactivation behavior is observed. (c) 1992 John Wiley & Sons, Inc.     

Effect of light and benzyladenine on dark-treated growing rice leaves (Oryza sativa). I. Changes in chlorophyll content and catalase activity

Light- and benzyladenine-induced reversal of the changes in chlorophyll content and catalase activity were studied in the attached first leaf of Oryza sativa L. cv. Bala, kept in darkness for different periods before maturation. Dark treatment caused a decrease in chlorophyll content and catalase activity at all times. Light treatment of dark-incubated seedlings at different periods before maturation reversed the dark-induced effect on chlorophyll content, catalase activity and dry weight and also caused a further rise in chlorophyll content compared to initial values. In darkness, the application of benzyladenine replaced the light effect in maintaining catalase activity. Chlorophyll content was also maintained by initially applied benzyladenine. Benzyladenine did not promote the photoinduced maintenance and increase in chlorophyll content and catalase activity at any time. Treatment with hydrogen peroxide, glycolate and amizol resulted in an accelerated chlorophyll breakdown and had varied effects on catalase levels. Chlorophyll decrease due to peroxide accumulation was to some extent reversible by benzyladenine, but the hormone had no effect on the peroxide-induced decrease in catalase activity. Development of catalase is light dependent. Benzyladenine stabilises the enzyme but has no effect on its synthesis.     

Survival and recovery of perennial forage grasses under prolonged Mediterranean drought: I. Growth, death, water relations and solute content in herbage and stubble

Swards of Dactylis glomerata cultivars (cvs) KM2 and Lutetia and of Lolium perenne cvs Aurora and Vigor were grown under full irrigation or prolonged summer drought (80 d) in a field experiment in the South of France.
After irrigation was withheld, leaf extension rates of all cvs fell by 90% within 9–12 d, and rapid scorching of laminae followed. Tiller mortality at the end of the drought was very different in the cocksfoot cvs (4% for KM2 and 76% for Lutetia) and intermediate (41%) for both ryegrass cvs. Following re-watering, rates of herbage regrowth were closely correlated with tiller survival. Measured minerals contributed c . 0·52 MPa to osmotic potential in all treatments, whereas water-soluble carbohydrates (WSC) contributed 0·25 MPa under irrigation and 0·46 MPa during drought.
There was no systematic difference between the two species for summer survival under severe drought, but large differences between the cocksfoot cvs. The traits most strongly associated with superior survival were: (a) a deep root system and greater water uptake at depth; (b) low water and osmotic potentials in surviving laminae, i.e. better tolerance to dehydration; (c) large pool-size of WSC reserves (fructans having degree of polymerization >4) in entire tiller bases (stubble); (d) low accumulation of proline in stubble; (e) rapid nitrogen uptake after rewatering.