Comparison Between Five Lysing Enzyme Preparations on Protoplast Formation in Aspergillus fumigatus

ID: 7

Group:

Fungal Molecular Biology

Prepared by:

Dr Mike Birch and (then Dr) Professor David Denning

Detail:

Five commercial lysing enzyme preparations were compared for their proficiency in generating protoplasts from A. fumigatus. Both total protoplasts and speed of formation were compared. Lysing enzyme from T. harzianum obtained from Interspex Ltd was the best, generating 2 x 107 protoplasts/g wet weight of mycelium in 2 hours.

Year prepared: 1998

Date uploaded: 1998-02-30

Introduction

A. fumigatus is a ubiquitous mould and an increasing cause of serious nosocomial infection in the immunocompromised patient [5]. Over recent years there has been a growing interest in genome based research using techniques such as RFLP analysis [2,4], PCR [1] and gene disruption [11]. A major obstacle is isolating high molecular weight DNA for use in such studies is the presence of a tough cell wall, components of which can co-purify and contaminate DNA preparations preventing further manipulation of the DNA [10]. Isolation of DNA from fungal protoplasts is one method which removes the need for extensive purification of DNA by CsCl gradients. Also this method is safer because it does not involve grinding mycelia with the subsequent production of mycelia dust.

Apart from DNA isolation protoplasts are useful in various fields of research. These include cell wall synthesis studies, genetic transfer using protoplast fusion, organelle studies and studies of biosynthetic pathways.

Factors affecting protoplast release are numerous [3] but of prime importance is the lytic enzyme (LE) used. Several commercial enzyme preparations have been used for protoplast and scheroplast production in fungi and yeast [6] with varying results.

In this comparative study 5 commercial lytic enzyme preparations were compared with respect to their ability to protoplast A. fumigatus mycelia. Enzymes were selected on the basis of availability, cost and on manufacturers reported specific activities (Table 1).

 

Table 1: Five commercial lysing enzymes used for protoplast formation in A. fumigatus AF245

Enzyme name Source Reported Activities Supplier Specific Activity
Lysing Enzyme T.harzianum Cellulase
Protease
Chitinase		 Sigma
Poole UK
Cat No L2265		 Not described
Lysing Enzyme T.harzianum β Glucanase
Cellulase
Protease
Chitinase		 InterSpex
Foster City USA
Cat No 0412-1		 Not described
Kitalase R.solani β Glucanase
Protease
Pectinase
Amylase		 ICN FLOW
High Wycombe UK
Cat No 153527		 Lytic 150000U/g
β Glucanase 2000U/g
Lysing Enzyme T.harzianum Cellulase
Proteinase
Xylanase
Chitinase		 ICN FLOW
High Wycombe UK
Cat No 152338		 β Glucanase 1500U/g
Glucanex Trichoderma spp β Glucanase Novo Nordisk Ferment Ltd
Neumatt CH
Cat No 367-5106		 β Glucanase 300U/g

Materials and Methods

InterSpex, Sigma, ICN and Kitalase are lytic enzyme preparations specifically produced for protoplast and spheroplast formation in fungi and yeast. Glucanex is a commercial enzyme preparation used in the clarification of wine hazes and has been reported to be useful in protoplast formation in Fusarium species [8].

A. fumigatus isolate AF245 isolated from a heart transplant patient with invasive aspergillosis was used in this study. Stored conidia were inoculated on to Potato Dextrose Agar in T75 cm2 tissue culture flasks and incubated for 7 days at 37°C or until confluent growth occurred. Conidia were harvested in 20 ml phosphate-buffered saline (PBS) with 0.1% Tween 80 by carefully disturbing mycelia with a microbiological loop. Harvested conidia were centrifuged at 1000 xg for 10 min and resuspended in 10 ml PBS/Tween and counted.

Erlenmeyer flasks containing 50 ml Vogel’s medium [13] were inoculated with 1×108 conidia and incubated at 37oC with shaking (200 rpm) for 21 hours. Mycelia was harvested by vacuum filtration through 4 layers of sterile muslin and blotted dry with filter paper. After weighing the mycelia was placed in a 50 ml flask and resuspended in protoplasting buffer, containing 2 mg/ml protoplasting enzyme, to a concentration of 40 mg mycelia per ml buffer [9].

Protoplasting buffer used with Sigma, InterSpex and ICN enzymes was 1.2 M MgS04 (pH 5.6). Protoplasting buffer used with Glucanex and Kitalase was 0.6 M KCl (pH 5.6). The choice of buffer for each enzyme preparation was based on manufacturers recommendations and preliminary experiments (data not shown). Mycelial suspensions were incubated at 33°C with gentle shaking (100 rpm). Formation of protoplasts was monitored by removing 2x 100μl aliquots of enzyme/mycelium suspension at hourly intervals and counting protoplasts manually using an Improved Neubauer Haemocytometer. Three counts were made on each 100μl aliquot removed. The whole experiment was conducted three times.

 

Results

The mean number of protoplasts produced at each time interval for each enzyme was calculated and plotted against time (Figure 1). Comparisons of protoplast production for each enzyme preparation were made by comparing the area under the curve (AUC) at each time interval. The mean number of protoplasts present at each time interval were compared using the multiple range test (modified least significant difference (LSD) procedure). This was carried out using the SPSS statistical package. Rate of protoplast production was calculated by determining the difference in formation of protoplasts between time intervals. Rates were compared using the modified LSD procedure again using the SPSS statistical package.

 

Figure 1: Protoplast production (per g wet weight of mycelia) from A. fumigatus strain AF245, over a six hour period, by five commercial enzyme preparations

Statistical analysis of AUC showed that the Interspex LE preparation produced the greatest number of protoplasts throughout the course of the experiment (p<0.01). In addition the Glucanex, Sigma and ICN LE preparations were superior to Kitalase (p<0.01).

The rate of formation of protoplasts was also compared. In the first hour Interspex LE was superior to all other preparations and Sigma LE superior to the other 3 preparations. Over hours 1-4 Interspex LE produced significantly more protoplasts than all other preparations (p<0.01). From hours 4-5 Sigma LE was superior to all others (p<0.01). In the 5-6 hour interval there was no significant difference in protoplast production between Interspex LE, Sigma LE, ICN LE and Glucanex (p<0.01). These preparations did differ significantly from Kitalase for the same interval.

 

Discussion

The chemical structure of the cell wall of A. fumigatus has recently been analysed [7] and was shown to consist of a glucan/chitin complex. Both alpha and beta glucan fractions were isolated with the beta glucans being composed of (1-3) and (1-6) linkages. Chitin was found to be linked to glucan by glucosamine residues.

The difference in the protoplasting ability of the different lysing enzyme preparations is interesting since 4 are derived from the same organism – Trichoderma harzianum. These lysing enzyme preparations from T. harzianum were superior to Kitalase from Rhizoctonia solani. The observed variation in protoplasting ability cannot be explained by differences in beta glucanase activity alone since Kitalase has the highest reported activity (2000 U/g) and was the least useful enzyme preparation. Other unreported ‘side’ activities must account for the observed variation. Thomas et al. [12] showed that the addition of chitinase greatly increased protoplast production from A. nidulans and A. fumigatus. It is likely that variation in chitinase activity between the 5 lysing enzymes accounted for the some of the variation in protoplast formation.

Other possibilities include increased lysis of protoplasts due to increased activities of protoplast degrading enzymes such as lipases or proteases.

This study has not addressed the relative merits of each lysing enzyme following growth in different media. Vogel’s medium was selected because it is one of the four defined media available which supports good growth of A.fumigatus and is composed of non complex constituents. It has been successfully used for the transformation of A.nidulans [14]. It is likely that growth medium for A.fumigatus has less influence on protoplasting efficiency than the lysing enzyme preparation and osmotica.

The cost of reagents is often a major influence in deciding on a particular product. In this study Glucanex was by far the cheapest preparation followed by Interspex lysing enzyme.

Acknowledgement

M. Birch was supported with grants from the North Western Regional Health Authority and the Fungal Research Trust.

References

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