HIGH-PRESSURE HOMOGENISATION OF BAKER’S YEAST FOR THE SELECTIVE RECOVERY OF INVERTASE

Wojciech Białas, Tomasz Jankowski
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poland

ABSTRACT

The process of yeast cell disruption using a high-pressure double-valve pilot-scale homogeniser has been investigated for the selective release of invertase. The first-order kinetics was applied for the determination of the release rate of invertase and total proteins. The release rate of invertase was higher than that of total proteins indicating this enzyme to be preferentially located outside the yeast cell membrane. It was found that the best conditions of the disruption process of the yeast cells were achieved for the operating pressure of 50 MPa and 10 passes of the cell suspension through the homogeniser. Increasing the disruption pressure above 50 MPa decreased the selectivity of invertase release, while extending the number of passes caused more shear damage to the enzyme.

Key words: cell disruption, homogeniser, yeast, invertase, location factor.

INTRODUCTION

Among many mechanical methods available for the disruption of cells for recovery of their intracellular enzymes, high-pressure homogenisation is preferentially used on large scale. Disruption in a high-pressure homogeniser is achieved by forcing a cell suspension through one or more valves once or in multiple passes. The disruption of cells in a homogeniser valve is caused by a number of hydrodynamic mechanisms such as shear stresses and impingement forces as well as turbulence and cavitations [17].

The efficiency of the cell disruption process depends on cell-wall mechanical properties [13], the location of the target enzyme within the cell [2], the operating pressure for the homogenisation and the number of passes through the homogeniser valve [5], and the type of mechanical device [10].

Although the APV-Gaulin homogeniser has attracted considerable attention, other types of high-pressure homogenisers are available for use in large scale cell disruption [8, 15]. Since the geometry and the gap size of the homogeniser valve can be different from type to type, they vary in performance for a particular cell disruption process. Therefore it is common practice in the biotechnology industry to optimise conditions of high-pressure homogenisation of a particular cell strain. This includes the choice of operating pressure, number of passes, and process temperature to obtain an efficient target protein release from the cells.

In the present study, the optimal conditions for disruption of Saccharomyces cerevisiae cells for the recovery of the intracellular enzyme invertase were considered. Homogenisation was performed using a NS2006L GEA pilot-plant high-pressure apparatus.

MATERIALS AND METHOD

Materials
For disruption studies baker’s yeast (Lasaffre Bio-Corporation) was suspended in a 0.1 M acetate buffer of pH 4.6 at a concentration of 29 g (dry weight)/l. Invertase (beta-D-fructofuranosidase, E.C. 3.2.1.26) from Saccharomyces cerevisiae obtained from Sigma-Aldrich was used without further purification. A standard solution of invertase at an activity of 0.8 µM/(min ml) was prepared in a 0.1 M acetate buffer of pH 4.6.

Methods
Cell disruption experiments were conducted using a double-valve NS 2006L pilot-scale homogeniser (GEA, Italy) equipped with an additional cooling coil mounted in a sample holding tank. Disruption pressure during the experiments ranged from 10 to 90 MPa. A total of 10 passes were performed at a constant flow rate equal to 80 l/h, while the circulating yeast suspension was cooled to 20°C before each pass. Samples of 2 ml were collected every second pass, cooled on ice, centrifuged at 10000 g at 4°C for 15 min.

The yeast cell disruption efficiency was estimated on the basis of invertase activity and the total soluble proteins released in the clear supernatant. For the assessment of invertase activity the 3,5-dinitrosalicilic acid method was used [9] and for the total proteins, the method of Lowry with bovine serum albumin as a standard was applied [16].

Separate experiments were conducted for the estimation of invertase stability during the homogenisation process. The conditions of these experiments were the same as for cell disruption but instead of a yeast cell suspension, a pure invertase standard solution was used.

RESULTS AND DISCUSSION

The effect of multiple passes through the homogeniser used in this work on the yeast cell disruption is shown in Fig. 1. One to three passes of a cell suspension through the homogeniser valves caused a rupture of the wall of the majority of the cells and a leakage of intracellular material. Further passes caused a subsequent disruption of all the cells into smaller fragments.

The release of invertase and total proteins from the yeast cells during the cell disruption process was described by the first-order kinetic relationship proposed by Hetherington et al [11] for a constant homogenisation pressure:

where R_max is the maximum obtainable protein release or enzyme activity after N passes through the homogeniser valve and k is release rate constant. Table 1 shows the release rates of invertase and the total protein during homogenisation of the yeast cells at different operating pressure calculated from the above relationship. It can be seen that the release rates of invertase and total protein were strongly influenced by the operating pressure. Thus, the higher the pressure, the more efficient was the homogenisation process. According to Hetherington et al [11] the relationship between the release constant and the disruption pressure has a form of an exponential equation k=k’P^a, where P is the pressure and k’ is a rate constant. The value of the exponent, a, is a measure of the resistance of a particular organism to disruption and also depends on its growth history [5]. To evaluate the pressure exponent for the yeast cells investigated in this study, a non-linear regression was carried out on a plot of k vs. the operating pressure P (Fig. 2).

The exponent, a, of the proposed equation had a value of 1.31 and 2.02 for invertase and total protein respectively. The lower value of the pressure exponent for the release of invertase than for that of total protein implies that for the same disruption pressure the enzyme was released relatively faster when compared to other proteins. This observation can further be confirmed by the results presented in Fig. 3, where for the chosen disruption pressure the amount of released enzyme was greater than the amount of total protein released. It should be added that this tendency was independent of the pressure applied.

Similar results showing different release rates of intracellular components were reported by other authors applying various cell rupture techniques [12, 14]. Their studies led to a general conclusion that enzymes located outside the cell membrane were released faster, while cytoplasmic enzymes were released at slower rates. Moreover, Umakoshi et al. [18] introduced a concept of a location factor allowing for the identification of location of a particular enzyme in the cell based on its release kinetics. The location factor was defined as follows:

where k_E is the release rate constant of the enzyme and k_T is the release rate constant of total protein. Thus, the enzyme is considered cytoplasmic, when the location factor is lower than one, and periplasmic, when the above ratio is greater than one. In terms of disruption technique used, the value of the location factor can indicate whether the particular equipment and operating conditions give selective enzyme release or not.

The values of the location factor of invertase obtained in this study are presented in Fig. 4. It can be seen that the values of the location factor were greater than one for the whole range of pressures applied. This confirms the observations of Burger et al. [3] and Arnold [1], who found this enzyme to be located in a soluble form outside the yeast cell membrane. It can also be seen in Fig. 4 that the location factor of invertase showed strong dependence on the operating pressure. A similar dependence was reported by Balasundram and Pandit [2], who showed that the location factor values of penicillin acylase, a periplasmic enzyme of E. coli constantly decreased when the homogenisation pressure increased in the range of 14 to 35 MPa. However, the relationship between the location factor and operating pressure shown in Fig. 4 appears to be more complex than that observed by the above authors. At a low operating pressure (10 MPa) the energy involved in a cell disruption process was probably too low to cause a significant rupture of the cell walls and a low release rate constant was observed. The maximum value of location factor of invertase, equal to 2.2, indicating the highest selectivity of the disruption process, was obtained for the pressure of about 40 MPa. A further increase of disruption pressure caused the location factor values to decrease, which implies that the cells were disrupted to a great extent and the cytoplasmic proteins started to release at a higher rate. It was interesting to note that the increase of pressure above 70 MPa did not change the value of the location factor. This suggests that for this operating pressure, maximum achievable disintegration of the cells has been obtained in the equipment used and a further increase of the pressure would not result in any significant change of this factor.

During the homogenisation processes proteins released from the cells can be subjected to extensive fluid forces which arise due to multiple pumping through a narrow homogeniser valves [7, 20]. The resulting hydrodynamic shear forces may cause damage to freely suspended enzymes, resulting in their denaturation and inactivation [4]. To study the effect of shear forces on a possible enzyme damage in our study, the solution of pure invertase was pumped through the homogeniser at the operating conditions similar to those used during the yeast cell disruption. However, comparing to the yeast cells disruption procedure, the time of treatment was extended to 30 passes (equivalent of 60 min.) and the lowest operating pressure of 10 MPa was not considered.

The results are presented in Fig. 5 as the relationship between the residual enzyme activity and the treatment time. Experimental data were analysed using a kinetic equation in the following form:

where A_R is the residual activity of invertase, k’’ is the inactivation rate constant of the enzyme, t is the time of treatment, and B is the constant. In addition to that, the values of half-lives (t₅₀) were determined from the logarithmic plots of the residual activity vs. time by linear regression analysis. Half-lives were defined as the time needed to reduce the enzyme activity to 50% of its initial value. The results of calculations are reported in Table 2. It can be seen that all operating pressures affected invertase activity but to a different extent. The data in Table 2 also show that the half-lives of invertase decreased when the homogenisation pressure increased. This means that the increase of the operating pressure produces more extensive shear rates which in turn may cause a serious loss of enzyme activity. However, half-lives showed in Table 2 were much longer than the time applied to disrupt the yeast cells in 10 passes. Using the above kinetic equation, the residual activities of invertase equivalent to 10 passes (R_A…N=10) normally used in the yeast disruption process were calculated. The numbers show that for two lower pressures (30 and 50 MPa) the loss of enzyme activity was lower than 10% and can be considered as negligible, while for the operating pressures of 70 and 90 MPa the effect of hydrodynamic shearing was more evident resulting in 17% and 34% enzyme inactivation respectively.

It is generally reported that most free enzymes are not susceptible to shear damage to any significant degree during high-pressure homogenisation [5]. However, there are few reports indicating the influence of the cell disruption method on denaturation of released enzyme. For example, during the disruption process in a bead mill, prolonged homogenisation caused a noticeable loss of enzyme activity [19]. Similarly, it was shown that the pressure had a greater impact on aminopeptidase loss than the number of passes, when Lactobacillus casei was subjected to the Microfluidizer treatment [6].

CONCLUSIONS

The observed variation of the location factor values for invertase as well as a possible loss of enzyme activity due to the hydrodynamic shear effects at different conditions of disruption suggest the need for a proper choice of process parameters and the extent of treatment in the applied equipment in order to obtain good selectivity and efficiency of the disruption process for a target enzyme. It was apparent from the present study that the best conditions for the disruption process of yeast cells were achieved for the operating pressure of 50 MPa and 10 passes of the cell suspension through the homogeniser. Increasing the disruption pressure above 50 MPa decreased the selectivity of invertase recovery, while extending the number of passes caused more shear damage to the enzyme.

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Accepted for print: 7.03.2007

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