Non-conventional yeasts as hosts for heterologous protein production.

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae.

Hydrolysis of used frying palm olein and sunflower oil catalyzed by porcine pancreatic lipase.

The enzymatic hydrolysis of frying used vegetable oils with different degrees of alteration were measured using porcine pancreatic lipase (acylglycerol acylhydrolase EC 3.1.1.3). Successive frying of potatoes significantly increased the level of total polar lipid content in the palm olein from 9.3 +/- 0.1 mg/100 mg oil to 26.4 +/- 0.3 mg/100 mg oil after 90 fryings, and from 4.0 +/- 0.1 mg/100 mg oil to 27.7 +/- 0.3 mg/100 mg oil in sunflower oil after 60 fryings. Triacylglycerol polymers, triacylglycerol dimers, and oxidized triacylglycerols also increased 37-, 7.9-, and 7.5-times in palm olein, respectively, and 56-, 22-, and 4.7-times in sunflower oil, respectively. However, diacylglycerols and free fatty acid levels related to hydrolytic alteration did not increase with the number of fryings in both oils. The substrate concentration in the reactor was determined by calculating the molecular weight of each oil showing a different degree of alteration. We compared the methodology used by us and that used by other authors. The results show that the methods are reproducible and that the values obtained are in concordance with theoretical values. The kinetic parameters apparent Michaelis-Menten constant (KMapp) and apparent maximum velocity of hydrolysis (Vmaxapp) were different in unused palm olein (5.1 +/- 0.7 and 166 +/- 7.6, respectively) than in sunflower oil (2.2 +/- 0.3 and 62 +/- 2.2, respectively). However, changes in KMapp and Vmaxapp were not related to the degree of alteration of the oils.

Comparative kinetic study of lipases A and B from Candida rugosa in the hydrolysis of lipid p-nitrophenyl esters in mixed micelles with Triton X-100.

(1) Lipases A and B from Candida rugosa catalyzing the hydrolysis of esters in micellar media have been characterized kinetically by studies on substrate specificity, rate equation forms and modeling of enzyme mechanisms. (2) The study on specificity revealed that both lipases are non-specific esterases with similar activity against lipid p-nitrophenyl esters micellized with Triton X-100. The slight difference was that lipase A has its maximum activity centered in the caprylate while that of lipase B is in the laurate. (3) Kinetic studies for both lipases were carried out with p-nitrophenyl laurate under three experimental conditions: (I) the molar fraction of substrate is fixed and the bulk concentration of substrate and Triton X-100 are varied; (II) the bulk concentration of substrate is held constant and the molar fraction of substrate and bulk concentration of Triton X-100 are varied; and (III) the bulk concentration of Triton X-100 is held constant but the bulk concentration of substrate and molar fraction of substrate are varied. (4) In case I, a similar Michaelis-Menten behaviour was observed with both lipases; the curve fitting gave kappcat/Kappm values of 3.0.10(5) and 5.6.10(5) s-1 M-1 for lipases A and B respectively. In case II, for both lipases the relationship between rate and the molar fraction of substrate required a fitting equation of 2:2 degree polynomial quotient. In case III, both lipases showed non-Michaelian behaviour with concave-up curves in the Eadie-Hofstee plot, a minimum degree of 2:2 in substrate concentration being detected for the rate equation. (5) The above results are interpreted in terms of the hypothesis that the mechanism of both lipases must include at least two different inputs for the molecule of substrate which would explain the quadratic terms observed in the rate equation.

Kinetic behaviour of a repressible acid phosphatase from the yeast Yarrowia lipolytica: a comparative study between the solubilized enzyme, the enzyme bound to cell-wall fragments and the enzyme bound to intact cells.

(1) The substrate specificities and types of inhibitors of a repressible acid phosphatase from the yeast Yarrowia lipolytica as solubilized enzyme, enzyme bound to cell-wall fragments and enzyme bound to the intact cell were found to be essentially the same. (2) A similar pattern for the activation of the enzymatic activity by ionic strength was found for solubilized enzyme, the enzyme in cell-wall fragments and the enzyme in intact cells. (3) v[S] studies with all three locations of the enzyme revealed non-linear Eadie-Hofstee plots with concave-up curves of the negative cooperativity type; these were correctly fitted with a rate equation of 2:2 degree polynomial quotient. In all cases, the same behaviour was obtained and no new kinetic properties were observed when the enzyme was bound to the cell-wall matrix with respect to the solubilized enzyme. (4) Inhibition by phosphate was characterized for the three locations of the enzyme by v[I] and v[S] studies. The same pattern of partial inhibition and non-Michaelian inhibition of 'non-competitive' nature was observed for all three forms. (5) The above results are interpreted in terms of the hypothesis that the cell wall of Y. lipolytica has a slight negative charge but behaves as a permeable matrix that does not lead to novel characteristics regarding the catalytic and regulatory properties shown by the enzyme molecule in free solution.

In vitro interaction between psychotropic drugs and alcohol dehydrogenase activity.

A series of CNS-stimulating and -depressant drugs have been studied for their in vitro interaction with horse liver alcohol dehydrogenase (ADH) activity. The depressant drugs studied included barbital, phenobarbital, thiopental, nitrazepam, chlorpromazine, sulpiride, clomethiazole, Li2CO3, diazepam, phenytoin, ethosuximide, morphine, and codeine. The stimulant drugs were theophylline, caffeine, amphetamine, imipramine, chlorimipramine, amitriptyline, and tranylcypromine. The results were as follows. First, ADH activity was inhibited by the action of chlorpromazine, tranylcypromine, imipramine, chlorimipramine, amitriptyline, sulpiride, amphetamine, codeine, ethosuximide, morphine, clomethiazole, nitrazepam, Li2CO3, theophylline, and phenobarbital, in descending order of inhibitory effect. Second, inhibition followed by activation of ADH activity was observed for imipramine and chlorimipramine. Third, activation of ADH activity was observed for phenytoin. Finally, the following drugs were not seen to exert any effect on ADH activity: barbital, thiopental, diazepam, and caffeine.

Kinetic properties of derepressible acid phosphatase from the yeast from of Yarrowia lipolytica.

(1) An acid phosphatase from depressed cells of the yeast form of Yarrowia lipolytica has been characterized kinetically by studies on specificity, inhibition, rate equation forms and modelling of the enzyme mechanisms. (2) The study on specificity revealed that the acid phosphatase is a rather unspecific phosphohydrolase that has similar activity on several different phosphate esters. A very weak transphosphorylating activity was also detected. (3) Among the reversible inhibitors, phosphate and vanadate were outstanding, whereas EDTA behaved as an activator. (4) v vs. [S] studies with o-carboxyphenyl phosphate as substrate show that the acid phosphatase of Y. lipolytica exhibits non-Michaelian behaviour, a minimum degree of 2:2 being detected for the rate equation in [S]. (5) The inhibition by phosphate and vanadate seems to have the same pattern of partial inhibition with a certain non-competitive nature, 1:1 being the minimum degree of the rate equation detected in (I).

The non-Michaelian action of thrombin on peptide p-nitroanilide substrates.

1. v([S]) kinetic data were obtained for the hydrolysis of the chromogenic substrates H-D-Phe-L-Pip-L-Arg-pNA (S-2238), H-D-Ile-L-Pro-L-Arg-pNA (S-2288), Tos-Gly-L-Pro-L-Arg-pNA (Tos-Ch-TH) and Cbz-Gly-L-Pro-L-Arg-pNA (Cbz-Ch-TH) by native human thrombin under different experimental conditions (Pip is pipecolyl, pNA is p-nitroanilide, Tos is tosyl and Cbz is benzyloxycarbonyl). 2. The data were fitted to rational functions of order 1:1, 2:2 and 3:3 by using non-linear regression. Discrimination between equations of different degree was made using the F-test. 3. In all, 24 curves were fitted. In 17 cases degree 2:2 was significantly better than degree 1:1 at a confidence level of 95%. In no case was any further significant improvement found with functions of degree 3:3. 3. Our results allow us to assert that native human thrombin is an enzyme that does not allow Michaelian kinetic behaviour when acting on chromogenic substrates. Instead, the empirically obtained steady-state data require mechanisms whose rate equation is at least of degree 2:2.

Kinetic study of the inhibition of the amidolytic activity of thrombin by benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581).

1. v([I]) data were obtained for the hydrolysis of the chromogenic substrate H-D-Phe-L-Pip-L-Arg-pNA (S-2238) by native human thrombin in the presence of the synthetic inhibitors Benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581). v([S]) data were also obtained in the absence and presence of fixed concentrations of each of the inhibitors. 2. Analysis of the kinetic data was based on the numerical fitting to rate equations of the polynomial quotient type of degree n:m using nonlinear regression methods. The discrimination between rate equations with different degrees was performed by application of the statistical F test. 3. Of eight v([I]) curves fitted, in six cases it was found that degree 1:2 was significantly better than degree 1:1 at a confidence level of 95% or higher; in no case was a significant improvement found with rate equations with a higher number of parameters. For the v([S]) data, of eleven curves fitted it was found that in nine cases degree 2:2 significantly improved degree 1:1 at confidence levels 99% and in one case at a level of 95%; no significant improvement was found with rate equations of higher degree for these data either. 4. Our findings allow us to propose that inhibition of the amidolytic activity of native human thrombin by benzamidine and I-2581 may be accounted for by mechanisms whose v([I]) rate equation will be a minimum of degree 1:2, thus implying a pure inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence concerning a possible steady state rate equation for E. coli alkaline phosphatase.

Steady state data was obtained for alkaline phosphatase over a wide range of experimental conditions using two substrates, four inhibitors, two modifiers and several pH, ionic strength and temperatures values. The data was fitted by rational functions of degree 1:1, 2:2 and 3:3 using a non-linear regression program and then the F-test was used to assess the goodness of fit. A proportion of the curves could only be fitted by 2:2 functions but many of them could be adequately fitted by 1:1 functions. No statistically significant improvement in fit occurred with 3:3 functions. Data was simulated using a computer program to see what sort of curves could be generated by a two sites mechanism proposed for alkaline phosphatase and this study showed it is difficult to detect cubic terms in this rate equation. It was concluded that alkaline phosphatase does not obey Michaelis-Menten kinetics. Rather, the steady state data require a mechanism of at least second degree but do not exclude a rate equation of third degree.

A mechanism for fibrin monomer aggregation based on a kinetic study.

A kinetic study was carried out of the aggregation of fibrin monomers under different reaction conditions. At either acid or base pH values, second-order kinetic processes were observed throughout the concentration range studied. At neutral pH (6.8 less than or equal to pH less than or equal to 7.3) the kinetics were of second order at fibrin monomer concentrations of less than 0.3-0.4 mg/ml, while at higher concentrations they changed to first order. Both the second order rate constants and the opacity of the fibrin gels are highly dependent on pH, ionic strength, the concentration of calcium ions and on temperature. A three-step reaction mechanism is proposed for the aggregation of fibrin monomers to explain the kinetic behaviour observed in the different reaction media.

Use of the F test for determining the degree of enzyme-kinetic and ligand-binding data. A Monte Carlo simulation study.

1. Initial-rate data were simulated for 13 representative enzyme mechanisms with the use of several distributions of rate constants in order to locate conditions leading to v([S]) curves in physiological ranges of substrate concentration. 2. In all, 420 sets of such v([S]) curves were generated with the use of several choices of substrate concentration range (two, three or four orders of magnitude), number of experimental points (10, 15 or 20), error on v (5-10%) and standard deviation on v (5-9%) in order to simulate experimental results in a number of possible ways. 3. Curve-fitting was carried out to rational functions of degree 1:1, 2:2, . . ., 5:5 until there was no statistically significant decrease in the sum of weighted squared residuals as judged by the F test at 95% and 99% confidence levels. 4. It was checked whether the non-linear regression program had located a good minimum in the sum of squares by also fitting the data with the correct values of parameters as starting estimates. 5. A similar procedure was adopted with 110 sets of binding data simulated for 11 models, and the F test was used to see if fractional-saturation data generated by a binding polynomial of order n could be adequately fitted by one of order m, m less than n. 6. From the 530 simulations the F test was successful in fixing the correct degree with a probability of 0.62 at the 95% confidence level, but this fell with increase in degree as follows: 1:1 (0.98), 2:2 (0.71), 3:3 (0.43) and 4:4 (0.34), the first numbers being the degree of the rate equation and those in parentheses referring to the 95% confidence level. 7. It made little difference whether the 95% or the 99% confidence level was consulted, as there were very few borderline cases. 8. The chance of detecting deviations from Michaelis-Menten kinetics, i.e. terms of at least second-order in a rate equation of degree n:n, n greater than 1, was estimated to be about 0.8. 9. The probability of the F test leading to a spurious result due to error in the data was found to be about 0.04. 10. The probability with which 4:4 mechanisms can lead to v([S]) plots with no, one, two or three turning points was computed, and it was established that there is a small but finite chance that the increase in degree that occurs in some mechanisms when ES in equilibrium EP interconversions are explicitly allowed for can be detected by the F test.

Negative cooperativity in alkaline phosphatase from E. col: new kinetic evidence from a steady-state study.

1. A study has been carried out on the steady-state kinetics followed by the alkaline phosphatase from Escherichia coli at different pH, temperatures, ionic strengths, phosphate concentrations and in the presence of the effectors such as Tris, NH4+--NH3 and CH3OH; p-nitrophenyl phosphate was used as substrate. 2. Contrary to what has generally been accepted, in most cases the enzyme follows non-Michaelian kinetics for a wide substrate concentration range, giving concave-down Lineweaver-Burk plots. Only at high phosphate concentrations (5 . 10(-3) M) and at high ionic strengths (2.0 M) is a linear Lineweaver-Burk plot obtained (Michaelian kinetics). 3. In order to analyse the kind of kinetics obtained, a non-linear regression fitting method was used to obtain rate vs substrate concentration equations as polynomial quotients of minimum degree with positive coefficients. 4. Most of the data obtained follows 2:2 degree type equations. 5. These results tend to suggest an idea of cooperativity rather than one of independence between the sites of the dimeric enzyme. A model is discussed for cooperativity between the sites with a wide concentration range giving concave-down Lineweaver-Burk plots.

Kinetic studies of the transphosphorylation reactions catalyzed by alkaline phosphatase from E. coli: hydrolysis of p-nitrophenyl phosphate and o-carboxyphenyl phosphate in presence of Tris.

1. Transphosphorylation of p-nitrophenyl phosphate and o-carboxyphenyl phosphate to Tris, has been studied at alkaline and acid pH. 2. The rate of release for all reactions products was Tris-dependent for both substrates, with a slight maximum for phenol at alkaline pH. These dependences have been analyzed from a mechanistic standpoint. 3. Individual constants of rate of a simple transphosphorylation mechanism have been determined. 4. At high Tris concentration (greater than 1.0 M) a slight competitive inhibition has been observed. 5. Inhibition in NH4+-NH3Cl buffer has been found at alkaline pH but not at acid pH. It would therefore seem that the non-protonated NH2 group of Tris is responsible for inhibition. 6. The results suggest the formation of complexes between Tris and the enzyme. Other possible alternatives are also analyzed.