Expression Of Recombinant Aspergillus Fumigatus Cellulases Biology Essay

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Five cellulolytic enzymes from Aspergillus fumigatus were expressed in Escherichia coli using the IPTG inducible pET 30 vector and checked for expression by IPTG induction. The samples obtained for all five enzymes after induction were subjected to SDS-PAGE electrophoresis, which revealed prominent bands for β-glucosidase (77 kDa), xylanase (27 kDa), endoglucanase (23 kDa) and cellobiohydrolase (60 kDa) while a faint band was observed for laccase (63 kDa). β-glucosidase showed particularly high level expression on denaturing polyacryl amide gel electrophoresis; this activity was associated with a 77 kDa protein. Using a sensitive colorimetric assay, based on the hydrolysis of p-nitrophenyl-b-D-glucopyranoside, it was studied for its kinetic properties. The optimal pH for high β-glucosidase activity was estimated to be in the range of 5.0-7.0 with maximum activity at pH 5.5 at an optimum temperature of 55ο C. The enzyme exhibited maximum activity after 15-20 minutes of incubation and was stable for 50 minutes. All high levels of expression allowed enzyme assays on whole cells without purification of β-glucosidase.

Keywords: Aspergillus fumigatus, β-glucosidase, cellulases, cellulose, Bioethanol

Abbreviations: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), β-glucosidase (β-gluc), laccase (lacc), xylanase (xylo), endoglucanase( endo), cellobiohydrolase (CBH)


Cellulases provide a key opportunity for achieving tremendous benefits of biomass utilization by enzymatic hydrolysis of cellulose (A.A. Sherief, 2010). The two main sources of current bioethanol production include corn and sugarcane which involve its production from glucose (Table 1). However, releasing it from cellulosic waste would both increase and economise its production. But currently, two main problems hindering their wide scale industrial application are low yield and high production costs (Xing-hua Li and Miao, 2009). Currently, the cost of producing these enzymes accounts for 40% of ethanol production costs. With fossil fuels rapidly depleting, there is a growing global urgency to optimise the production of cellulolytic enzymes to make cellulosic ethanol a reliable source of fuel.

Cellulose is a fibrous, insoluble, crystalline polysaccharide (Xing-hua Li and Miao, 2009). It is the most abundant carbohydrate polymer on earth, being the major polysaccharide component of plant cell walls and is mainly composed of repeating D-glucose units linked by β-1, 4 glucosidic bonds (Xing-hua Li and Miao, 2009). Cellulases are a specific group of enzymes that catalyse the conversion of cellulose to soluble sugars and glucose (Fig 1) (Bhat, 1997) since cellulose is highly resistant to enzymatic attack. It is thus an essential group of enzymes for facilitating the depolymerisation of cellulose into fermentable sugars (Xing-hua Li and Miao, 2009). These enzymes have been found to have important applications in different industries such as textile, fuel, brewery, paper and pulp, waste management and food industries (Supp. Table1). The synergistic action of atleast three enzymes (Fig 1), namely cellobiohydrolase (CBH), endoglucanases (Endo), and ,β-glucosidases (Bhat, 1997) is required to breakdown rigid cellulose fibres. The exoglucanases (mainly CBH) and endoglucanases hydrolyze cellulose to the

Insoluble Cellulose


Cellodextrins Cellobiose

Endo β-gluc


Cellobiose Glucose

Fig 1: Possible synergistic action of cellulases to facilitate enzymatic degradation of cellulose (Bhat, 1997)

disaccharide cellobiose (Supp. Table 3). Cellobiose is in turn hydrolyzed into simple glucose subunits by a β-glucosidase (Day, 1982). The extracellular cellulose system in fungi is comprised of three main components : 1) endoglucanases or CM-cellulases (E.C., 2) exoglucanases or cellobiohydrolases (E.C. and 3) β-glucosidases (E.C. (M. Umar Dahot ). This makes fungi an ideal system to isolate the desired group of cellulases for cellulose breakdown.

Trichoderma reesei (Supp. Table 2) is one of the most common fungal strains (model fungal organism) being used for the production of industrially relevant enzymes like proteases, cellulases, amylases, hemicellulases (Buchert J, 1998) . Among cellulases, it has been proven to secrete high amounts of cellobiohydrolase and an endoglucanase complex including a variety of endoglucanases. However, low β-glucosidase activity in the secreted complex is posing to be the rate limiting factor in industrial utilisation of these enzymes (Eduardo A. Ximenes, 1996) since the role of β-glucosidase to breakdown cellobiose into glucose subunits (Supp. Table 3) is indispensable for the production of bioethanol. Another major obstacle in their industrial utilisation is the low rate of hydrolysis (Erick J. Vandamme, 1982).

Aspergillus fumigatus is a thermotolerant strain of fungi with higher growth and cellulose production rates than the mesophilic fungi Trichoderma reesei. It has been reported that A. fumigatus strains are excellent decomposers of crude cellulosics including jute, filter paper, hemi-cellulose, pulp and straw (Erick J. Vandamme, 1982). They have been shown to have optimal cellulose activity at higher temperatures (50-55οC), which makes them more adapted to harsh conditions of industrial processes. The use of higher temperatures in ethanol production might offer the benefit of increasing reaction rates by eliminating contaminating micro-organisms thereby increasing the efficiency of glucose production

Table 1: Current sources of fuel ethanol




Fossil fuels

A few dominant countries with depleting oil reserves

Non-renewable/ rapidly depleting sources


Mainly USA

Pressure on food crops. Not feasible and economical in the long run/high cost of raw material


Mainly Brazil

High cost raw material

Lignocellulosic biomass

Can be utilised worldwide

Renewable and sustainable- Currently, a very expensive process, 40% of production costs is due to cellulases.

The overall aim of the project is to use tobacco chloroplasts for high level expression and containment of recombinant proteins for the degradation of cellulose, ultimately for the production of bioethanol. The main objective of present work was to characterise the expression of recombinant A.fumigatus proteins in E.coli. This would provide information on their properties before expression in chloroplasts. This required establishing conditions for optimal induction of recombinant proteins using SDS-PAGE to verify the appearance of recombinant proteins of the correct size. The best possible growth conditions were therefore tested by varying media and temperature for incubation of bacterial cells. This was followed by enzyme assay for β-glucosidase activity where optimum temperature and pH were determined to facilitate maximum enzymatic activity.

2. Materials and methods

2.1. Materials

Acrylamide (30% w/v acrylamide, 0.8% w/v bis-acrylamide) was obtained from Protogel, national diagnostics, USA. p-NPG was obtained from Glycosynth, England, Gel protein marker-Precision plus was obtained from Bio-rad Laboratories and the DC assay kit was also purchased from Bio-rad Laboratories.

2.2. Protein extraction from pET30 vectors in pLysS BL21 E.coli cells

E.coli containing the required foreign protein from frozen stock cultures were streaked out on fresh LB plates containing 100 µg ml-1 kanamycin (10 mg ml-1 stock) (Melford, UK) and 30 µg ml-1 chloramphenicol (10 mg ml-1 stock) (Sigma, UK). Plates were incubated for 16 hours at 37οC. Single colonies from plates were used to inoculate 5mls of LB with 100 µg ml-1 kanamycin and 30 µg ml-1 chloramphenicol in 10 ml falcon tubes and incubated for 14-16 hrs in water bath (shaker incubator) at 37οC at 250rpm.On the next day, 1ml of the 5 ml cultures was used to inoculate 250 ml conical glass flasks containing 50mls of LB with 100 µg ml-1 kanamycin and 30 µg ml-1 chloramphenicol, which were incubated at 37οC at 250 rpm in a water bath (shaker incubator). To harvest cells at an optical density of an absorbance at 600nm (A600) of 0.4, 1ml of the 50 ml culture was taken aseptically and measured spectrophotometrically after every 30 mins and subsequent samples were taken until A600 of 0.4 was obtained. Following this, the culture was split into two conical flasks, each containing 25 mls of growing culture. 1mM isopropyl-β-D-thiogalactopyranoside (IPTG, BioGene) was added to one flask to induce expression and the other flask was used as a control. The flasks were incubated for two hours under the same conditions described above and 0.5 ml of culture was harvested in 1.5 ml sterile centrifuge tubes after every 30 mins to prepare protein samples. Alongside, 1ml of culture was also collected at these 30 min intervals to check for A600 value for each sample, which would be required to prepare protein samples for gel loading as per the calculation in section 2.3.

The same procedure was repeated for protein extraction for all five cellulases. Endoglucanase and cellobiohydrolase were also cultured in NZCYM media keeping all the other conditions the same for one set of experiments and varying only the temperature to either 37οC or 30οC.

2.3. Preparation of harvested protein samples for polyacrylamide gel electrophoresis

0.5 mls of culture was removed after every 30 mins from the growing culture and transferred to sterile 1.5 ml centrifuge tubes. All centrifuge tubes were spun at 14,000 rpm for 1 min, the supernatant was discarded and the pellets were immediately frozen in liquid N2. At the end of 2 hours (after IPTG induction), after harvesting and centrifuging all the samples (4 in total from induced culture and one from uninduced culture), the pellets were stored at -80οC. The 0.5 mls culture pellet from the 1.5 mls centrifuge tubes were resuspended in 1x sample buffer (10% glycerol, 5% βmercaptoethanol, 3%SDS, 0.0625 M Tris pH 6.8, 0.1% bromophenol blue) according to calculation below to get equal gel loadings:

(Final A600)/0.4 x 50µl (1x sample buffer) = volume of sample buffer to be added

Due to the presence of βmercaptoethanol, the sample buffer was added in the fumehood. Subsequently, the samples were placed in a boiling water bath for 5 mins after which they were vortexed briefly. Protein samples were either used to load the gel on the same day or stored at -20οC for future use (upto 3-4 weeks).

2.4. Polyacrylamide gel electrophoresis for protein fractionation

For Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), the Protean III system (Bio-rad, UK) was used. Separating gel was used in different concentrations for different enzyme strains. The percentage acrylamide used for the separating gel was 10%, 12% or 7.5% and for the stacking gel was 4%. The separating gel was comprised of 0.375M Tris-HCl pH8.0, 0.1% w/v SDS, 33% v/v of 30% acrylammide/Bis (Protogel, national diagnostics, USA), 0.05% w/v ammonium persulfate (APS, BDH,UK), 50% v/v TEMED (SIGMA-ALDRICH, USA) in sterile de-ionised H2O . The stacking gel was composed of 0.252 M Tris-HCl pH 6.8, 0.1% w/v SDS, 13% acrylamide/Bis, 0.05% w/v (APS), 50% v/v TEMED in sterile distilled water. The gel was run in running buffer (0.025 M Trizma base, 0.192 M glycine (Fischer Scientific, UK), and 1% w/v SDS pH 8.6) in the fume hood. 5 µl of the protein marker Precision Plus all blue protein marker (Bio-rad laboratories) was loaded and 10 µl of each of the protein samples were loaded. The gel was run for approximately 2 hrs at 100 V. It was then removed from the apparatus and stained in coomassie brilliant blue (Expedeon protein solutions, UK) for nearly 60 mins, while shaking on an orbital shaker. It was then rinsed in water and subsequently scanned to obtain an electronic image.

2.5. Determination of enzyme activity

β-glucosidase activity was determined using the method provided by CALZYME Laboratories Inc. The modifications made in the procedure were as follows; the enzyme was incubated till 20 mins and A400 was measured after every 5 mins. A temperature range of 37- 65οC was considered instead of only 37οC as suggested in the protocol. A pH range of 4.5-7.5 was considered as opposed to suggested pH optima at 5.0. The optical density was measured at different wavelengths (300, 350, 380, 390, 400, 405 and 420 nm) to determine the optimal wavelength for activity measurement (appendix 2). This was done with 0.02 mM and 0.01mM concentrations of 4-Nitrophenol. Following this, a calibration curve was prepared using 7 sequential dilutions of 4-Nitrophenol (Sigma Aldrich, UK) ranging from 0.05-1.6 µmol (Supp. Fig 3) at 400 nm, to determine the activity of β-glucosidase after the assay. The method of assay is based on the reaction of p-Nitrophenol, which is formed during the reaction and is determined spectrophotometrically at 400nm.


p-NPG + H2O D- Glucose + Alcohol

Fig 2: Breakdown of p-NPG by β-glucosidase to produce glucose

For the assay, fresh E.coli cultures were grown and induced under the same conditions (as described above) and 2 mls of culture was obtained in 2ml centrifuge tubes and cell pellets were obtained. These were re-suspended in 1.2 mls 0.1 M acetate buffer [8.2 %w/v in sterile distilled water, CH3COONa (BDH,UK)] pH 5.5 and 0.8 mls of freshly prepared 0.02M p-NPG (0.6% w/v) was added to each tube. The tubes were immediately put in a water bath at 55οC and incubated for 20 minutes. 0.5 ml of the sample was removed after every 5 mins and added to pre-labelled 1.5 ml centrifuge tubes containing 0.5mls of 0.2 M Na2CO3 (BDH,UK) to stop the reaction. After collecting all the samples, their OD was measured at A400nm.

One unit of enzyme activity was defined as the amount of enzyme that catalyses the formation of one µmol of p-nitrophenol min-1. The final activity measurements for pH range and activity at 55ο C and pH 5.5 were performed three times.

E. coli Streaked plates

1ο selection- single colony incubation

(in LB with antibiotics at 37οC)

2οselection- shake flasks (in LB with antibiotics at 37οC)

Sample isolation

Protein expression on SDS-PAGE


Fig 3: Scheme for cell growth and sample isolation.

Fresh cells were obtained for the activity assay following

the same procedure.

2.6. Effect of temperature on enzyme activity

The optimum temperature for β-glucosidase assay was determined as described above. Temperature stability was determined by incubating β-glucosidase at various temperatures in 0.1 M acetate buffer, pH5.5 and sampling after every 5 mins. Optimum enzyme activity was determined (as described above) at 55οC.

2.7. Effect of pH on enzyme activity

The optimum pH for the assay was measured as described previously. pH stability was determined by resuspending the β-glucosidase pellets in 0.1M acetate buffers, pH 4.5-7.5, at 55οC for 20 mins. 0.5 mls of sample was removed from the water bath after every 1 min and added to pre-labelled sterile 1.5 ml centrifuge tubes (Supp. Fig 4). Three consecutive sets of samples were taken in one experiment. The reaction was stopped by adding an equal volume (0.5mls) of 0.2 M Na2CO3. Enzyme activity was determined by measuring the OD at A400 (as described above).

2.8. Total protein determination using DC Protein Assay

The Bio-rad DC Protein Assay is a colorimetric assay for protein concentration following detergent solubilisation. The assay is similar to the well documented Lowry assay and is based on the reaction of protein with an alkaline copper tartarate solution and Folin reagent. 10 mg/ml BSA (Sigma-Aldrich, USA) stock was used as the protein standard. Five serial dilutions ranging from 0.125mg/ml-2mg/ml protein were prepared from the stock. This was used to draw a standard curve (appendix 7), where absorbance was measured at 750nm. 2ml of fresh E.coli-β-glucosidase culture was pippeted in a 2ml centrifuge tubes and centrifuged for 60 sec at 14,000rpm in a bench-top centrifuge. The supernatant was discarded and the pellet was resuspended in 2ml autoclaved water. Immediately, 50µl of the cell sample and 50µl of each of different BSA solution concentrations were pippeted into clearly labelled centrifuge tubes. 250µl reagent A (alkaline copper tartarate solution was added to each tube followed by 2ml of reagent B (dilute Folin Reagent). The tubes were incubated at room temperature for 15 mins, following which their OD was read at 750 nm.

3. Results

3.1. Bacterial growth curves

The scheme in fig 3 shows the steps leading to cell harvest for optical density measurement to check for their growth rate. At first, all the bacterial strains containing recombinant plasmids were grown in LB at 37οC and for every culture; the OD was measured at 600 nm. Readings were noted after 30 mins right from inoculating the shake flasks and used to plot the curves (Figs 4, 5 and 6). On plotting the curves, it was observed that the curve corresponding to the induced cells was lying a little below the curve for uninduced cells (Fig 4), this can be due to cell lysis during protein expression. In order to induce expression in endoglucanase and cellobiohydrolase containing transformants, different culture conditions were tried by varying the temperature to 30οC and changing the enzyme medium to NZCYM. Growth curves for cellobiohydrolase and endoglucanase are shown in fig 5 and fig 6 respectively.


Fig 4: The growth curve for β-gluc with induced and uninduced cells at 600 nm (representing the lag and log phase) and the secondary Y-axis, depicting total protein content 0.41mg/ml.Similar curves were obtained for Endo, Xylo, Lacc and CBH under the same conditions (in LB at 37οC)


Fig 5: The growth curve for CBH in NZCYM media at 30οC. It represents the lag and log phase, stationary could not be shown since the cells were harvested during exponential phase itself. Similar curves were obtained for strains expressing endo under the same conditions.


Fig 6: The growth curve for the strain expressing Endo in LB at 30οC. A Similar curve was obtained for a strain expressing CBH in NZCYM at 30ο C.

3.2. Analysis of recombinant protein expression by SDS-PAGE

Coomassie staining was used to visualise the strong protein bands for induced β-glucosidase and xylanase samples, and a faint band for laccase but not for endoglucanase and cellobiohydrolase under similar conditions (LB at 37οC). These bands were obtained at the expected sizes of 77, 27 and 63 kDa respectively, compared to their uninduced controls (Fig 7). This shows that the proteins for β-glucosidase, xylanase and laccase can be successfully expressed at the above mentioned culture conditions. To determine if the level of protein expression can be induced by varying the culture conditions for endoglucanase and cellobiohydrolase strains, first the media was altered from LB to NZCYM and cultures were grown at 37οC. When still no bands could be observed for both, the temperature was then altered from 37οC to 30οC for both the media. Fairly strong bands could then be observed for both at the respective sizes of 23 and 60kDa (fig 7). This shows that proteins accumulate to different levels in E.coli and are expressed at different levels. Table 2 shows a summary of molecular weights of protein bands as observed for all five strains.

A) B) C)

D) E) F) G)

Fig 7: pET 30(b) inducible expression of CBH, β-gluc, endo, xylo and lacc in E.coli BL21 PLysS. The figure shows the total protein from uninduced (U) and induced (I) E.coli, expected size of protein labelled on right side. (A) CBH, uninduced and induced on 12% SDS-PAGE cultured in Luria Broth (LB) at 30οC. (B) β-gluc, uninduced and induced on 10% SDS-PAGE cultured in LB at 37οC. (C) β-Gluc on 7.5% SDS-PAGE. (D) Endo, uninduced and induced on 10% SDS-PAGE cultured in LB at 30οC. (E) Xylo, uninduced and induced on 10% SDS-PAGE cultured in LB at 37οC. (F) Lacc, uninduced and induced on 7.5% SDS-PAGE cultured in LB at 37οC.(G) Precision plus molecular marker (P+), 10-250 kDa , obtained from Biorad used to estimate molecular weight.

Table 2: Observed molecular weights of isolated cellulases (from SDS-PAGE analysis)


Molecular weights (kDa)





Cellobiohydrolase (CBH)






3.3. pH optima

1ml cell samples from fresh growing cultures were resuspended in acetate buffer with pH ranging from 4.5-7.5 and the activity was determined as described above. The enzyme showed considerably high activity in the range of 5.0-7.0 (Fig 8), but the activity recorded at pH 4.5 was low compared to the expected results since β-glucosidase from T. reesei has been reported to have optima at pH 4.8. High activity in a broad pH range indicates that on being transformed in tobacco chloroplast, the enzyme might retain partial activity since the plant chloroplast stroma have an alkaline environment with a high pH of 8.0.

Fig 8: pH optimum for β-gluc activity was observed at pH 5.5 when the assay was performed for the pH range of 4.5-7.5. The samples were incubated for 15 mins at 55οC and three consecutive samples were collected for each value at an interval of 1 min.

3.4. Temperature optima

Temperature optimum for β-glucosidase activity was recorded at 55οC (Fig 9), when the fresh cell culture samples, for both induced and uninduced cells were resuspended in acetate buffer pH 5.5 and incubated for 15 mins in water baths set at different temp (Table 4). A drop in activity was noted on increasing the temperature beyond 55οC, but still more than 70% of the enzyme activity was retained when compared to the maximal activity at pH 5.5 and 55οC (Tables 4 and 5). Although, an optimum temperature could not be determined for uninduced cells since their values observed were random and did not show a definite trend. But still, maximum activity was observed at 55ο C and showed just around 50% activity compared to the induced cells.

Fig 9: Temperature optimum for maximum β-gluc activity was observed at 55ο C. Absorbance was measured at 400 nm for all the sets of experiment in the range of 37οC to 65οC at pH 5.5. Activity trend was observed for both, induced and uninduced cells.

3.5. Protein content and specific activity

The amount of protein loaded per well was calculated using the DC Protein Assay. A calibration curve of BSA standard (Supp. Fig 7) was plotted (after 15 minutes of incubation). The enzyme sample was assayed under the same conditions and consequently, amount of protein was calculated in 5µl sample. Approximately 21µg of protein was estimated to be loaded per well. The amount of protein present in 1 ml culture at an A600 of 0.4was estimated to be 0.41mg (Table 3).

Table 3: Summary of enzyme activity parameters


Total Act.

Total Protein (1ml culture)

Specific Activity

(Tot. Act/Tot. protein)



Opt. Temp.


0.0820 μmol min-1


0.200 μmol mg-1min-1



Table 4: Summary of recombinant β-gluc activities measured at different temperatures, keeping pH constant at 5.5. All the samples were incubates at specified conditions for 15 minutes. All the values represent an average of three readings. Amount (µmol) of product released under stated conditions is also indicated.



Abs (I) (400nm)

µmol (p-nitrophenol)

Abs (UI) (400nm)

µmol (p-nitrophenol)
















0.4329 (max. Activity)















Table 5: Summary of recombinant β-gluc activities measured at different pH values, keeping the temperature constant at 55οC. All the samples were incubates at specified conditions for 15 minutes. All the values represent an average of three readings. Amount (µmol) of product released under stated conditions is also indicated.



Abs (I) (400nm)

µmol (p-nitrophenol)

Abs (UI) (400nm)

µmol (p-nitrophenol)

55 οC






55 οC






55 οC



0.4329 (max. Activity)



55 οC






55 οC






55 οC






55 οC







A. fumigatus has proven to be an effective producer of a mixture of all five essential cellulases- β-glucosidase, CBH, endoglucanase, xylanase and laccase. As shown previously, maximum induction was observed in β- glucosidase as opposed to other four cellulases, β- glucosidase and xylanase showed high protein expression compared to endoglucanase, laccase and cellobiohydrolase expression which implies that this could potentially offer a solution to the problem of lack of production of β-glucosidase from Trichoderma sp(A.A. Sherief, 2010) . Also, in order to induce endoglucanase and CBH strains, an attempt was made to modify the growth conditions to check if their expression could be enhanced. Hence NZCYM media was used as a variation since it consists of Casein digest, yeast extract and casamino acids which provide the necessary nutrients and cofactors required for excellent growth of recombinant strains of E. coli. However, still altering growth conditions did not enhance endoglucanase and cellobiohydrolase expression in NZYM at 37ο C, therefore temperature was also varied to 30οC and as a result, some protein expression bands could be observed for both the strains on growing them in LB at 30οC. The reason for no strong induction could be due to variation in protein stability or limited translation due to the codons present.

To check for the cellulolytic activity of the crude β-glucosidase, p-NPG assay was performed. According to literature, the optimal activity for β-glucosidase should have been observed at pH 5.0. However, for the given enzyme from A. fumigatus, it was found out to be at pH 5.5 and 55οC. This can be attributed to the thermotolerant nature of A. fumigatus since β-glucosidase was found to possess considerable activity even at 65οC. Hence, the isolated enzyme from fumigatus is relatively more thermostable which will facilitate their use in different industrial processes(N. Ait, 1979). A high temperature tolerance could also have the benefit of increasing reaction rates thereby reducing the time to hydrolyse cellulose, which inturn will increase the efficiency of glucose production. This will further add to the viability of bioethanol in future by further reducing the production costs. It was also noted that the enzyme is highly thermostable since it retained more than 70% of its activity after 50 minutes of incubation. However, if it is desirable to use this enzyme to supplement the cellulase from T. reesei (pH 4.8 and 50°C) in industrial saccharification, the A. fumigatus enzyme would be active. At pH 5.5, its activity was only 18% higher than the activity observed at pH 5.0 and there was a 24% increase in activity at 55οC compared to the activity at 50οC. Highest cellulose activity from A. fumigatus has previously been observed at 55ο C(Erick J. Vandamme, 1982). Moreover, a higher temperature and pH optima could have other industrial significance since the pH and temperature optima for Thermoactinomyces sp. β-glucosidase have been observed to be at pH 6.5 and 50 to 55°C (Hagerdal, 1979); for Huumicola insolens, pH 5.0 and 50°C (Yoshioka, 1980) (Sternberg, 1977). To further check the effectiveness of isolated β-glucosidase on cellulose, an activity assay on cellobiose as the substrate can be further performed.

Sonication for cell lysis did not have any significant effect on increasing the enzyme activity and neither did the use of proteases. This was unexpected as the cell lysis should have released the protein in the buffer, thereby increasing its activity. The β-glucosidase was not purified in this work. Purification of the enzyme would allow more detailed analysis of its kinetic properties.

Even though the pH optima for β-glucosidase is lower than that of the chloroplast stroma, where the cellulases have to be expressed, it still showed considerable activity in the range of 5.0-7.0, with 70% of its activity still retained at pH 7.5. This indicates that these cellulases when transformed in tobacco chloroplasts may be active and can be successfully expressed. So far, only two of the five main cellulases from A. fumigatus have been isolated and studied in detail : A 12.5 kDa major endoglucanase from A. fumigatus had shown to have a pH optimum of 4.8 and a temperature optimum of 60οC (parry, 1983), and Bauer et al. (2006), had expressed xylogalacturonase from A. fumigatus in Pichia pastoris. However, the exact activity levels of the enzyme were not determined(Bauer, 2006).