JOURNAL OF ;bMIR&LOGICAL

ELSEVIER
Journal of Microbiological Methods 22 (1995) 193-205

Permeabilization of the methylotrophic yeast Pichia pinus for intracellular enzyme analysis: a quantitative study
Tiina Alamie*,
Instit&e of Molecular and Cell Biology. Received 11 October 1994; revision received

Aiki Jarviste
Tartu, Estonia 3 January 1995 12 December 1994; accepted

University of Tartu, Riia 23, EE2300

Abstract

A quantitative in situ assay of methanol oxidizing enzymes in permeabilized suspensions of the methylotrophic yeast Pichia pinus is described. Activities of the intracellular enzymes alcohol oxidase, formaldehyde dehydrogenase and formate dehydrogenase were readily measured in cell suspensions treated with 0.1% surfactant, digitonin or cetyltrimethylammoniumbromide (CTAB) for 15 min. Recovery of enzyme activities from the permeabilized cell suspension was higher than from disrupted cells. The permeabilizing ability of surfactant was dependent on its concentration, being highest at 0.1%. Cell permeabilization caused a marked decrease in dry weight and protein content of cells whereas no significant immidiate leakage of the intracellular enzymes alcohol oxidase, formaldehyde dehydrogenase and formate dehydrogenase was observed. However, 20-h storage of permeabilized suspension at 8C resulted in marked leakage of dehydrogenases but not Iof alcohol oxidase from the cells. Addition of CTAB at 0.01% and digitonin at 0.01% and 0.1% to the cell-free extract for 1 h did not inhibit formaldehyde dehydrogenase and alcohol oxidase, whereas incubation with CTAB at 0.1% for the same time reduced ,activity of these enzymes to 62% and 37% from the initial, respectively. Properties of alcohol oxidase (K, towards methanol and substrate specificity) in permeabilized cell suspensions were similar to these measured in cell-free extract. Treatment of cell suspension with surfactants reduced the viability of cells dependent on the type of surfactant and its concentration.
Keywords: Alcohol yeast; Pkhia pinus

oxidase;

Cell permeabilization;

Enzyme

analysis;

Methylotrophic

* Correrponding author. Phone: +372 7 420223; Fax +372 7 435440; E-mail: talamae@ebc.ee. Abbreviations: CTAB, cetyltrimethylammoniumbromid; dig, digitonin; Aox, alcohol oxidase; HCHO dh. formaldehyde dehydrogenase; HCOOH dh, formate dehydrogenase; DHA. dihydroxyacetone; GAP, glyceraldehyde-3-phosphate; GSH, reduced glutathione; MW, molecular weight; FPLC, fast protein liquid chromatography. 0167-7012/95/$09.50 0 1995 Elsevier SSDI 0167-7012(95)00007-O Science B.V. All rights reserved

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1. Introduction

Enzymatic reactions in yeasts are usually studied in cell-free extracts. The preparation of cell-free extracts is laborious and needs disruption of cells, which destroys the integrity of cells and may cause inactivation of enzymes. Therefore, cell permeabilization has been recommended as an alternative method for the study of intracellular enzymatic reactions. Detailed studies on yeast cell permeabilization for enzymatic analysis have been published for Kfuyverornyces fragilis and Saccharomyces cerevisiae. These yeasts have been permeabilized with Triton X-100 [l], CTAB [2], digitonin [3], toluene [4-51, and by freezing and thawing [1,6]. These studies have shown that enzyme activities measured in permeabilized cells might more precisely reflect the in vivo situation than activities measured in cell-free extracts. We have isolated and studied Pichia pinus mutants with glucose repression-insensitive synthesis of methanol-oxidizing enzymes. As preparation of cell-free extracts for enzymatic assay of numerous isolates was too laborious we decided to use cell permeabilization. Though digitonin-treated suspensions of methylotrophic yeasts have in some cases been used for measurement of intracellular enzymatic activity [7], systematic studies on using permeabilized cells for the quantitative assay of enzyme activities in methylotrophic yeasts were lacking. In methanol-yeasts, cell permeabilization has mostly been used for qualitative evaluation of enzyme activities on colonies permeabilized with either chitosan, digitonin or cation M2, cetyldimethylbenzylammonium chloride [&lo]. To develop a suitable method for permeabilization of the methylotrophic yeast Pichia pinus for enzymatic analysis, recovery of activities of methanol-oxidizing enzymes with different molecular weight and intracellular localization were compared in surfactant-treated and disrupted cells. The effect of different concentrations of surfactant on cell permeability and on activity of enzymes in cell-free extracts will be shown.

2. Materials

and methods

2.1. Materials Methylotrophic yeast Pichia pinus MH4 was obtained from the Institute of Technical Chemistry, Leipzig. Chemicals used: digitonin (Fluka), cetyltrimethylammoniumbromid, CTAB (Chemapol), NAD + and horseradish peroxidase (Reanal). Other chemicals used were of analytical grade. 2.2. Growth conditions and harvesting Yeasts were maintained on YEPD (g/l: yeast extract-2, peptone-20, glucose-20) agar slants. Cells were grown at 28C under batch conditions on mineral medium (11) with continuous aeration. Methanol (0.3% v/v) was used as a carbon source.

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Cells were harvested in the exponential phase of growth by centrifugation (6000 g, 15 min, 4C) and washed twice with 50 mM potassium phosphate (KP) buffer, pH 7.4. The cell pellet was used for permeabilization or stored frozen (-1sOC) until disruption. 2.3. Disruption of cells and preparation of cell homogenates Frozen cells were disrupted using a Hughes press and suspended in KP buffer. This suspension was used for enzymatic analysis either directly (suspension of disrupted cells), or after centrifugation at 15 000 g for 30 min (in this case supernatant was analyzed as the cell-free extract).
2.4. Permeabilization qf cells

One gram (wet weight) of washed cells was suspended in 10 ml of KP buffer containi:ng 0.1% digitonin or 0.1% CTAB, w/v. If permeabilizing effect of different concentrations of surfactants was studied, O.Ol-0.5% of surfactant was used. The cells were incubated in this solution for 1.5 min at room temperature and occasionally stirred with a glass rod. At the end of the incubation, the cells were pelleted and washed twice with KP buffer by centrifugation. Washed cells were suspended in 15 ml of KP buffer and maintained in an ice-bath with a magnetic stirrer. The resulting suspension was used for the assay of the enzymes either directly or after dilution with KP buffer. 2.5. Dry weight and protein determination Dry weight content of the suspensions was measured by weighing of cells on membrane filters after their drying at 104C for 24 h. Protein was determined according to Lowry [12], using bovine serum albumin as a standard. Suspensions of disrupted or permeabilized cells were treated with 1 N NaOH at 100C for 30 min to solubilize the proteins before applying the Lowry procedure. 2.6. Determination of the cell viability Viability of permeabilized cells was determined by plating diluted suspensions before and after permeabilization onto the agar plates containing methanol (2% v/v), and counting colonies after the incubation on Petri plates for 3 days at 28C. 2.7. Enz,yme assays The alcohol oxidase activity was measured by following the amount of formaldehyde formed from methanol during the incubation of the enzyme preparation (suspension of permeabilized cells, suspension of disrupted cells or the cell-free extract) with 40 mM methanol in a KP buffer [13]. The total volume of the incubation mixture was 8 ml. Samples (I ml) were withdrawn at regular

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intervals and the reaction was stopped by heating the samples at 100C for 5 min. Formaldehyde in the samples was estimated by addition of 1 ml of Nash reagent and further incubation for 10 min at 60C [14]. If the substrate specificity of alcohol oxidase was studied, the activity was measured by following the formation of H,O, as described by Eggeling and Sahm [15], but using o-dianisidine instead of ABTS as a chromogen. Formaldehyde and formate dehydrogenases were assayed by following appearance of NADH as a function of time. The incubation mixture for formaldehyde dehydrogenase contained: 50 mM KP buffer, pH 7.4; NAD 0.33 mM, reduced glutathione 0.6 mM and formaldehyde 3 mM [16]. If formate dehydrogenase was assayed glutathione was omitted and 33 mM sodium formate was added instead of formaldehyde. All measurements were performed at room temperature (22C). The amount of enzyme preparation was chosen to give changes in absorbance of less than 0.2001min. Measurements were repeated until at least three coinciding absorbance changes per minute were recorded. One unit (U) of enzyme activity was defined as the amount of enzyme transforming 1 pmol of substrate per minute.

3. Results and discussion

3.1. Use of 0.1% CTAB and 0.1% digitonin for permeabilization of Pichia pinus cell suspensions for the assay of methanol-oxidizing enzymes

The following enzymatic reactions were studied in permeabilized alcohol HCHO + H202 CH,OH + O2 HCHO HCOOH + GSH + NAD+
formate formaldehyde dehydrogenax

cells: (I) + NADH (2)

> GS-CHO
dehydrogenase >

+ NAD+

CO,

+ NADH

(3)

As presented in Table 1, surfactant-treated Picha pinus cells exhibited high enzymatic activity that was close to or even higher than the activity of the disrupted preparation. We did not use enzymatic activities of cell-free extract (supernatant of disrupted cells) as a reference to evaluate the permeabilizing efficiency of surfactants, because different enzymes were unevenly distributed between the cell-free extract (supernatant) and the pellet fraction as shown in Table 2. is needed to To analyze enzyme activities in situ, cell permeabilization overcome the permeability barrier of the cell membrane to substrates and products of enzymatic reactions. In this work the activity of alcohol oxidase was assayed by following formaldehyde production from methanol (see Materials and methods). Methanol and formaldehyde penetrate cellular membranes by diffusion [17]. Therefore cell permeabilization should not be obligatory for their passage through membranes. However, intact cells exhibit only minor alcohol oxidase

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Table 1 Enzyme <activities (units) recovered from 100 mg (dry weight) of methanol-grown exposed to different methods of treatment
Cell

P. pinus cells

Enzyme activity; units (pmollmin) Alcohol oxidase P DW P DW 11.4 100% 12.8 112% 16.2 142% 0.4 3% P 0.05 0.04 0.06 0 DW 1.8 100% 1.3 72% 1.7 94% 0 0% 30.8 100% 46.7 152% 44.6 145% 0.9 3% 0.33 0.40 0.56 0.01 Formaldehyde dehydrogenase Formate dehydrogenase

preparation

Suspension of disrupted cells Digitonintreatedb CTABtreated Intact cellsd

0.88 1.46 1.44 0.03

P, enzyme activity (units) per mg of protein; DW, enzyme activity (units) per 100 mg dry weight of cells. a Frozen cells were disrupted using a Hughes press. After disruption the cell debris was suspended in KP buffer and used as the enzyme preparation. The activity of the disrupted cell preparation was taken as 100% and compared to the enzyme activities recovered from the same amount of cells subjected to permeabilization. b.cCells were permeabilized with digitonin (0.1%) or CTAB (0.1%) for 15 min. and twice washed with KP buffer (see Materials and methods) and used as the enzyme preparation. Washed :suspension of intact (untreated) cells was used as the enzyme preparation. Table 2 Distribution of the cellular protein content and enzyme activity (Aox, alcohol oxidase; HCHO dh, formaldehyde dehydrogenase; HCOOH dh, formate dehydrogenase) in preparing the cell-free extract Volume (ml) Suspension of disrupted cells Supernata& Pellet 4.0 3.4 0.6 Protein (mg) 18.0 100% 4.7 26% 13.3 74% Units (fimol/min) Aox 15.8 100% 5.3 34% 10.5 66% HCHO dh 5.9 100% 4.3 73% 1.6 27% HCOOH dh 0.9 100% 0.5 56% 0.4 44%

a Prepared as described in Table 1. b Suspensicn of disrupted cells (a) was centrifuged at 16 OOOg +4C for 30 min and enzyme activities in the supertratant were measured. Enzyme activities (units) in the pellet are calculated by subtracting the amount of enzyme units in the supernatant from the amount of units in the suspension of disrupted cells.

activity (Table 1). In methylotrophic yeasts alcohol oxidase resides together with dihydroxyacetone synthase and catalase in membrane-bound organelles, peroxisomes [18]. Formaldehyde produced in an alcohol oxidase reaction can be used for biosynthesis and trapped inside the peroxisome:

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HCHO

+ xylulose-Sphosphate

nu* synt=se DHA

+ GAP .

However, it can also diffuse into the cytosol and bind reduced glutathione to be further oxidized by formaldehyde dehydrogenase to generate energy (see reaction 2 above). Formaldehyde production and utilization by the cell is well coordinated and thus no marked leakage of formaldehyde from the cell occurs. Our data show that after addition of methanol to the permeabilized suspension, formaldehyde immediately diffuses out, enabling in situ measurement of alcohol oxidase activity It could be proposed that rapid leakage of by formaldehyde production. formaldehyde from permeabilized cells is caused by shortage of low molecular weight trapper molecules (xylulose-5-phosphate and glutathione) from the cell. Activities of formaldehyde dehydrogenase and formate dehydrogenase were assayed by following production of NADH from NAD. Pyrimidine cofactors cannot penetrate the intact cell membrane [2] and therefore cell permeabilization must follow in situ NAD reduction. In spite of that some formaldehyde dehydrogenase activity (up to 3%) could in some cases be detected in suspension of untreated i.e. intact cells (Table 1) being probably caused by lysed cells present was in the cell population. However, no activity of formate dehydrogenase detectable if untreated suspensions were studied, because its activity in methanolgrown P. pinus is always much lower than that of formaldehyde dehydrogenase [ill. CTAB has not been used for permeabilization of P. pinus before. The comparison of enzyme activities in CTAB- and digitonin-treated cells demonstrated that alcohol oxidase activity in CTAB-treated cells was 87% from that of the digitonin-treated cells while activities of formaldehyde dehydrogenase and formate dehydrogenase were 121% and 151%, respectively (Fig. 1). Thus CTAB treatment should be recommended for the assay of dehydrogenases. 3.2. Properties of alcohol oxidase in permeabilized
cells

Permeabilized cells could be considered to resemble the natural environment of the intracellular enzymes more closely than the cell-free extracts commonly used to measure enzyme activities. We studied properties of alcohol oxidase in P. pinus permeabilized cell suspensions and in cell-free extract. K, values of alcohol oxidases for methanol in different methylotrophic yeasts range from 0.67-3.1 mM [19-211. According to our results, the K, value of alcohol oxidase for methanol in CTAB-permeabilized cells of P. pinus was about 1 mM, and in cell-free extract, about 3 mM (data not shown). Alcohol oxidase of methylotrophic yeasts contains bound formaldehyde that can diffuse out of the enzyme that results in an increase of alcohol oxidase affinity for methanol [21,22]. Therefore, differences in K, values between permeabilized cells and cell-free extract could be due to enhanced diffusion of formaldehyde from permeabilized cells. Substrate specificity of alcohol oxidase in permeabilized cells and in cell-free extract was similar (Table 3) and comparable to data presented in the literature for alcohol oxidases of methylotrophic yeasts [13, 191. Thus, alcohol oxidases in permeabilized cells and

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199

160 140 120 8 100 80 60 40 20 0 Aox HCHOdh 67ea

151+6

HCOOHdh

Fig. 1. Comparison of the permeabilizing efficiency of CTAB and digitonin. Activities of methanol oxidizing (enzymes (mean 2 SD) in suspensions of Pichia pinus treated with either 0.1% digitonin or 0.1% CTAB for 15 min were compared. Exponentially growing cells from three independent batch cultivations were permeabilized with 0.1% of surfactant. Activity of the enzyme in digitonin-treated suspension was taken as 100%. Aox, alcohol oxidase; HCHO dh, formaldehyde dehydrogenase; HCOOH dh, formate dehydrogenase. Table 3 Substrate specificity of alcohol oxidase in digitonin-treated Alcohol 300 mM Methanol Ethanol Allylalcohol Propanol Butanol Relative activity (%) Digitonin-treated 100 64 41 35 21 cells Cell-free extract 100 61 44 42 30

cells and in cell-free extract of P. pinus

in cell-free extracts behave similarly. Therefore, our results are in good agreement with data of Gowda et al. [2] on alcohol dehydrogenase in S. cerevisiue permeabilized cells. 3.3. Leakage of enzymes from P. pinus cells treated with
digitonin for 15 min
U.l%

CTAB

or 0.1%

Treatment of yeasts with surfactants causes leakage of intracellular molecules, including, enzymes, from the cell [2,3,23]. Therefore, dry weight and protein content of cell suspensions were determined before and after the permeabilization of cells. Supernatants obtained after the pelleting and washing of permeabilized cells with KP buffer were pooled (further referred to as washing buffer) and analyzed for protein content as for enzymatic activity. Treatment of cells with surfactants reduced their dry weight by 30% and

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Table 4 Dry weight and protein content of P. pinus suspensions before and after the treatment for 15 min with 0.1% digitonin or 0.1% CTAB. Data are recalculated to represent 100 mg (dry weight) of cells subjected to permeabilization Permeabilizer 0.1% Dry weight before Digitonin CTAB 100 100 (DW) mg DW loss % 29 30 Protein before 35 35 mg after 32 31 Protein loss % 9 12

after 71 70

protein content by 9-12% (Table 4). If Can&da bozdznzz cells were treated with 0.1% cation M2 for 2 h, they lost 46% of their cellular protein [23]. Compared to [23] the average protein loss in our experiments was much lower, probably due to the shorter treatment time. FPLC analysis of washing buffer for proteins showed that mainly small proteins (MW below 60 000) were released from the cells (data not shown). Though the majority of released proteins probably originate from the cell membrane, leakage of intracellular enzymes has also been reported. Marked release of formaldehyde and formate dehydrogenases but not of alcohol oxidase from C. boidinii treated with Cation M2 for 2 h has been shown by Sakai and Tani [23]. We also detected some activity of dehydrogenases in the washing buffer of surfactant-treated cells. The highest level was recorded for formate dehydrogenase activity in the case of digitonin-treatment, that ranged up to 10% from the intracellular level of the enzyme (data not shown). Molecular weights of formaldehyde dehydrogenase and formate dehydrogenase in methylotrophic yeasts are 80 000 and 74 000, respectively [16]. These values are only slightly higher than the molecular weight of larger proteins detected in the washing buffer of permeabilized cells. Therefore, cytosolic dehydrogenases of P. pinus were expected to leak from the cells during the long-time storage of the permeabilized suspension as has been previously reported for P-galactosidase in CTAB-treated Kluyveromyces luctis [24]. To control this hypothesis, distribution of different enzymes between intra- and extracellular space after the 20 h storage of the suspension was studied. As presented in Fig. 2 the majority of the dehydrogenases of formaldehyde and formate indeed diffuses out while a peroxisomal enzyme alcohol oxidase (octamer with combined molecular weight about 800 000) remains inside the cell. Considering the differential leakage of enzymes from surfactant-treated Pichia pinus cells, surfactant treatment could be used for separation of alcohol oxidase from dehydrogenases in the course of purification of these enzymes from P. pinus as it has been used for alcohol oxidase purification .. from Cundidu bozdzmz [22]. 3.4. Effect of surfactant on enzyme activity in cell-free extract As enzymes leak out during the permeabilization of the suspension they could be affected by the surfactant. Effect of CTAB and digitonin on enzymes was studied in P. pinus cell-free extracts using alcohol oxidase and formaldehyde

. . ..

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201

Aox dig

HCHO dh dig

HCOOH dh dig

AOX CTAB

HCHO dh CTAB

HCOOH dh CTAB

Fig. 2. Distribution of alcohol oxidase. formaldehyde dehydrogenase and formate dehydrogenase (%) between Intra- and extracellular space after 20 h of storage in KP buffer at 8C in CTAB- or digitoa-,treated (dig) P. pinus suspensions. The stored suspension was centrifuged at 6000g for 15 min and enzyme activities were measured in the pellet (permeabilized cells) and in the supernatant. Total activity (100%) of the enzyme recovered from the suspension after storage was calculated by summarizing enzyme units detected in the pellet and in the supernatant. Aox, alcohol oxidase: HCHO dh. formaldehyde dehydrogenase; HCOOH dh, formate dehydrogenase.

dehydrogenases as examples. Fig. 3 shows that CTAB at 0.1% inhibited activity of both alcohol oxidase and formaldehyde dehydrogenase. Addition of 0.1% of digitonin and 0.01% of either digitonin or CTAB had no marked effect on protocol the cell suspension was routinely enzymes. In our permeabilization treated ,with 0.1% of surfactant for 15 min and after that cells were twice washed with KP buffer. Taking into account the high recovery of enzymatic activity from permeabilized cells (Table 1) and good stability of enzymatic activity of permeabilin,ed suspension (90-100% from the initial level after the 20 h storage at SOC), we consider that permeabilizing agents can be effectively removed by washing of treated cells. Therefore no significant inhibition of enzymatic activity takes place during treatment and keeping of permeabilized suspension in spite of the fact that dehydrogenases partly diffuse out. 3.5. Comparison of perrneabilizing ability of different concentrations of
surfactants

Permeabilizing ability of different concentrations of CTAB was evaluated by activity of alcohol oxidase and formaldehyde dehydrogenase in permeabilized suspensions. As shown in Fig. 4 activities of both enzymes were highest in cells treated with 0.1% of CTAB. Thus, a 0.1% concentration of the surfactant that has been recommended for permeabilization of Saccharornyces cerevisiae and Kluyveromyces fragilis [3,6], was also appropriate for Pichia pinus.

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HCHO dh

Methods 22 (1995) 193-205

100 bp

-L -t_ -

CTAB. CTAB,

0.1% 0.01%

80
60 40 20 0 0 15 30 time, 45 min 60

.&
I$

Dig. 0.1% Dig, 0.01%

Treatment

Aox 100 90 80 70 E8 --Dig, 2 ;: 20 10 0 0 15 Treatment 30 time, 45 min 60 -t_ Dig, 0.01% 0.1%

CTAB. CTAB,

0.1% 0.01%

M 3

Fig. 3. Effect of digitonin and CTAB on alcohol oxidase and formaldehyde dehydrogenase in cell-free extract. Digitonin or CTAB were added to the cell-free extract of methanol-grown cells at concentrations 0.1% or 0.01%. After the incubation of the extract with surfactant at room temperature for the time indicated, the activities of alcohol oxidase and formaldehyde dehydrogenase were measured. Specific activity of the corresponding enzyme in the untreated cell-free extract was taken as 100%.

3.6. Viability of permeabilized

cells

As a rule, permeabilized cells have mostly been considered to be non-viable [25-261. We determined the viability of P. pinus cells before and after the treatment for 15 min of cells with lower (0.01%) and higher (0.1%) concentrations of CTAB and digitonin. Treatment of cells with CTAB reduced their viability more rapidly and severely than treatment with the same concentration of digitonin (Fig. 5). However, as part of the cell population could still survive even after 15 min treatment with surfactant, the conditions of surfactant treatment can be varied to obtain cells with the expected permeability and/or viability.

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1.8
1.6 .s ; : 1.4 1.2

~ -Aox
i HCHO dh

g
3 s

1 0.8

,s 0.6 .> z 0.4

0.2 0 0
0.1

0.2
CTAB, %

0.3

0.4

0.5

Fig. 4. Activities of alcohol oxidase (Aox) and formaldehyde dehydrogenase (HCHO dh) in P. pinus suspensions permeabilized with different concentrations of CTAB. One gram of washed methanolgrown ceils was treated for 15 min with 10 ml of CTAB in KP buffer at concentrations indicated and enzyme amctivities in suspensions were measured.

--

dig 0.01% digO.I% CTAB 0.01% CTAB 0.1%

10

15

treatment time, min

Fig. 5. Viability of I? pinus cells treated with different concentrations of digitonin or CTAB. Diluted suspensions of washed methanol-grown cells were plated before and after the treatment of the suspension with surfactants for the time indicated, and colonies were counted after the incubation.

3.7. Short protocol for permeabilization analysis

of methylotrophic yeasts for enzyme

For the in situ quantitative assay of methanol-oxidizing enzymes in yeasts we recommend the following protocol. One gram of wet cells should be treated with 10 ml of 0.1% digitonin or CTAB for 15 min, washed thereafter two times with buffer and kept in buffer on an ice-bath under continuous stirring during the experiment. In the case of smaller or higher than 1 gram amount of cells,

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appropriate portions of 0.1% surfactant in KP buffer should be used to give a cell-to-surfactant ratio of 1: 0.01 (w/w). The permeabilized suspension can be maintained in a cool place for at least 20 h without significant loss of enzymatic activity.

4. Concluding

remarks

Our results showed that permeabilization of cells is a feasible method for the study of enzymatic activity of methylotrophic yeasts: 0.1% CTAB- and 0.1% digitonin-treated cells exhibited even higher activities of enzymes of methanol oxidation than disrupted cells. Enzymatic activities are usually measured in cell-free extracts (supernatants of disrupted cells). Our data show that enzymes of methanol oxidation were unevenly distributed between the cell-free extract and pellet fraction. Therefore, according to our data in situ measurement of enzymatic activity using permeabilized cells most appropriately describes the in vivo situation, and could be recommended for the study of enzyme activities in methylotrophic yeasts.

Acknowledgments

The authors wish to thank Dr. Anne Kahru for revising the manuscript and Dr. Jaan Simisker for valuable comments on the paper.

References
[l] [2] Miozzari, G.F., Niederberger, P. and Hinter, R. (1978) Permeabilization of microorganisms by Triton X-100. Anal. Biochem. 90, 220-233. Gowda, L.R., Joshi, M.S. and Bhat. S.G. (1991) Permeabilization of bakers yeast by cetyltrimethylammonium bromide for intracellular enzyme catalysis. Enzyme. Microb. Technol. 13. 154-1.57. Gowda, L.R., Joshi, M.S. and Bhat, S.G. (1988) In situ assay of intracellular enzymes of yeast (Kluyveromyces fragilis) by digitonin permeabilization of cell membrane. Anal. Biochem. 175. 531-536. Murakami. K., Nagura, H. and Yoshino. M. (1980) Permeabilization of yeast cells: application to study on the regulation of AMP deaminase activity in situ. Anal. Biochem. 105, 407-413. Serrano, R., Gancedo, J.M. and Gancedo, C. (1973) Assay of yeast enzymes in situ. Eur. J. Biochem. 34, 479-482. Seip, J.E., and Di Cosimo, R. (1992) Optimization of accessible catalase activity in polyacrylamide gel-immobilized Saccharomyces cerevisiae. Biotechnol. Bioeng. 40, 638-642. Sibirny, A.A., Titorenko, V.I., Gonchar, M.V., Ubiyvovk, V.M., Ksheminskaya, G.P. and Vitvitskaya, O.P. (1988) Genetic control of methanol utilization in yeasts. J. Basic Microbial. 28, 293-319. Eggeling, L. and Sahm, H. (1980) Direct enzymatic assay for alcohol oxidase, alcohol dehydrogenase and formate dehydrogenase in colonies of Hansenula polymorpha. Appl. Environ. Microbial. 39, 268-269.

f3]

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[8]

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of Microbiological Methods 22 (1995) 193-205

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[9] Sibirny, A.A., Titorenko, V.I., Efremov, B.D. and Tolstorukov, 1.1. (1987) Multiplicity of mechanisms of carbon catabolite repression involved in the synthesis of alcohol oxidase in the methylotrophic yeast Pichia pinus. Yeast 3, 233-241. [lo] Sakai, Y., Sawai, T. and Tani, Y. (1987) Isolation and characterization of a catabolite repressioninsensitive mutant of a methanol yeast Candida bmdmr A5, producing alcohol oxidase in glucose-containing medium. Appl. Environ. Microbial. 53, 1812-1818. [ll] Alamae, T., Teugjas. H., Soom, J. and Simisker, J. (1985) A partly resistant to catabolite repression mutant of the yeast Pichia pinus assimilating methanol. Microbiologiya 54, 634-640 (in Russian). [12] Lowry, O.N., Rosenbrough. A., Farr. K. and Randall, K. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. [13] Tani Y., Miya, T., Nishikawa, H. and Ogata, K. (1972) The microbial metabolism of methanol. Part I. Formation and crystallization of methanol-oxidizing enzyme in a methanol-utilizing yeast, Kloeckera sp. No 2201. Agr. Biol. Chem. 36, 68-75. [14] Nash, T. (1953) The calorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55, 416-421. [ 151 Eggeling. L. and Sahm, H. (1978) Derepression and partial insensitivity to catabolite repression of the methanol dissimilating enzymes in Hansenula polymorpha. Eur. J. Appl. Microbial. 5, 195-202. [16] Shiitte. H.. Flossdorf, J.. Sahm, H. and Kula, M.-R. (1976) Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida bozhnu. Eur. J. Biochem. 62. 151-160. [17] Stein, W.D. (1967) The Movement of Molecules Across Cell Membranes, p. 77, New York: Academic Press. [18] Sulte:, G.J.. Harder, W. and Veenhuis, M.(1993) Structural and functional aspects of peroxisomal membranes in yeasts. FEMS Microbial. Rev. 11, 285-296. [19] Couderc. R. and Baratti, J. (1980) Oxidation of methanol by the yeast. Pichia pasroris. Purification and properties of the alcohol oxidase. Agric. Biol. Chem. 44, 2279-2289. [20] Dijken, J.P., van, Otto, R. and Harder, W. (1976) Growth of Hansenula polymorpha in a methanol-limited chemostat. Arch. Microbial. 111, 137-144. [21] Bystrykh, L.V.. Romanov, V.P., Steczko, J. and Trotsenko, Y.A. (1989) Catalytic variability of alcohol oxidase from the methylotrophic yeast Hansenula polymorpha. Biotechnol. Appl. Biochem. 11, 184-192. [22] Sakai, Y. and Tani Y. (1988) Simple purification and improvement of stability of alcohol oxidase from a methanol yeast Candida boidinii S2, by forming an enzyme-azide complex. Agric. Biol. Chem. 52, 227-233. [23] Sakai, Y. and Tani, Y. (1988) Production of formaldehyde by detergent-treated cells of a methanol yeast Candida boidinii S2 mutant strain AOU-1. Appl. Environ. Microbial. 54, 485-489. [24] Joshi, M.S., Bachhawat, N. and Bhat. S.G. (1989) Stabilization of cetyltrimethyl-ammonium bromide permeabilized yeast whole cell lactase. Biotechnol. Lett. 11, 349-352. [25] Lusta, K.A., Maslakevich, P., Sysoev, O.V. and Trotsenko, Yu. A. (1993) Immobilization of digitonin-treated methylotrophic yeast Hansenula polymorpha. Prikl. Biokhim. Mikrobiol. 3, 381-388 (in Russian). [26] Felix, H. (1982) Permeabilized cells. Anal. Biochem. 120. 211-234.