There is an increasing trend towards using enzymes for catalyzing biotransformations 12. In view of their high costs (as compared to chemical catalysts), reusable forms of enzymes, called immobilized enzymes, are often used 34. Immobilization is also sometimes accompanied by greater stability of enzymes during storage or operational stability. Immobilization generally consists of linking an enzyme to a solid matrix or entrapping it in a gel. Insoluble enzyme aggregates produced by extensive chemical cross-linking of enzyme dissolved in a solution have been suggested as an alternative approach for obtaining stable and reusable enzyme preparations 56. In recent years, it has been shown that chemical cross-linking of enzyme precipitates produces more robust biocatalysts. These have been called cross-linked enzyme aggregates (CLEAs) 789.

An interesting feature of the CLEAs is that these preparations do not require extensive purification of the enzyme activities. In this respect, CLEAs differ from the CLEC™, another form of enzyme aggregates prepared by chemical cross-linking of enzyme crystals 10. It has been shown that it is possible to form a CLEA that can catalyze more than one reaction. Thus, a CLEA may catalyze a sequence of reactions. Such CLEAs have been called Combi-CLEAs 811. Recently, we suggested that it should be possible to extend this concept to Combi-CLEAs catalyzing non cascade reactions. Prepared from porcine pancreatic acetone powder, a CLEA was described which had lipase, alpha amylase and phospholipase A₂ activities 11. Such multipurpose CLEAs would allow "one biocatalyst for many unrelated biological activities". In the present work, we show that this concept is especially useful in the context of crude/commercial preparations of microbial origin. In as much as enzymes are increasingly isolated from microbes, this work shows that a single biocatalyst preparation from microbial sources could catalyze several biotransformations/bioconversions of industrial relevance.

The system chosen is a well-known commercial preparation called Pectinex™ Ultra SP-L that is used in the food processing industry for hydrolyzing pectin 12. Our earlier work had shown that this preparation is also rich in xylanase and cellulase activities 1314. A multipurpose CLEA with substantial activities of pectinase, xylanase and cellulase was prepared and characterized. The other two activites, xylanase and cellulase, also have well known and extensively documented applications in biotechnology 1516.

The general protocol for the preparation of CLEAs consists of precipitating the enzyme activity by adding salt or an organic solvent 79. This is followed by addition of cross-linking reagent, which is generally glutaraldehyde. Figure 1 shows that n-propanol precipitated all three of the enzymatic activities (viz. pectinase, xylanase and cellulase) completely. Figure 2 shows the effect of varying glutaraldehyde concentration. Following the conditions described in the literature 911, 4 h of cross-linking time at 4°C was chosen for this study. As the results show, even a 5 mM glutaraldehyde concentration was good enough to result in CLEA, which retained 100% of all the three enzyme activities originally present in the solution.

The pH optima for the three activities in the CLEA were more or less same as those compared to free enzyme activities. The pH optima were broad (at 4.5 for pectinase and 5.5 for xylanase and 5 for cellulase activities) and agreed with the literature value 171819. In the case of cellulase activity, CLEA showed pH optima at 5.5 as compared to 5 observed and reported for free enzyme (Data not shown as figure).

The temperature optima also did not reveal significant changes. The temperature optima observed for free enzymes agreed well with the reported value 171819. The pectinase temperature optima was 50°C for both free enzyme and CLEA. Free xylanase showed a temperature optimum of 50°C. CLEA showed broad temperature optima in the range of 50–55°C. Free cellulase has a temperature optimum of 50°C. CLEA showed a broad temperature optima in the range of 50–55°C (data not shown).

Table 1 summarizes the value for Michaelis-Menten parameters obtained with the three activities with free enzyme solution and CLEA. Values of V_max were found to increase in the case of all three activities measured in CLEA, whereas K_m values remained more or less unchanged upon CLEA formation. While not much data is available regarding these changes in the kinetic parameters upon CLEA formation, the trends observed in the present case agree with those obtained with CLEA formed from pancreatic acetone powder 11. It may be mentioned that cross-linking is known to improve marginally the catalytic efficiency of enzyme 20. Immobilized enzymes generally show increases in K_m values especially when enzyme activities are assayed with high molecular weight substrates. In the present instance, K_m values did not increase in a significant manner. Considering that CLEA was formed with just 5 mM glutaraldehyde, perhaps the enzyme aggregates had a very open structure and mass transfer remained as easy as with free enzyme.

Table 2 shows the remarkable thermostabilization of the enzymes present in the preparation. In all three of the cases, half-lives have increased upon CLEA formation. Cellulase activity was most thermostable and its thermoinactivation was measured at 70°C. Pectinase was least stable and its thermoiactivation was measured at 50°C. The largest increase in stability was in the case of pectinase in which the half-life increased from 17 to 180 minutes.

The Scanning electron micrograph (SEM) of this CLEA preparation is shown in Figure 3. The CLEAs were found to have diameter in the range of 15–30 μm. The size of various CLEAs (and their clusters) has been reported to be in the range of 0.1 μ to 100 μm 7.

Figure 4 shows the reusability of CLEA for all the three enzyme catalyzed reactions. In all the three cases, CLEA could be reused three times without any loss of any of the enzyme activities. After this, slow decline in the activities in all three cases started.

To conclude, the present work outlines the preparation of a multipurpose robust biocatalyst. The biocatalyst preparation is more thermostable and reusable. The multipurpose biocatalyst can be used for bioconversions (such as hydrolysis of pectin, xylan and cellulose) of considerable biotechnological relevance.

Xylan and polygalacturonic acid were purchased from Sigma Chemical Co., St. Louis, USA. PectinexTM Ultra SP-L (a commercial preparation of pectolytic enzymes from a selected strain of Aspergillus niger) was a kind gift from Dr. J.S.Rao, Novozymes, Bangalore, India. All other chemicals used were of analytical grade.

MNG suggested the idea about multipurpose CLEA, identified the source of multiple enzyme activities and was actively involved in the discussions of the results. AS optimized the conditions for preparation of CLEA. SD carried out the characterization of CLEA, SEM studies of CLEA and enzyme assays. All authors jointly drafted, proof read and approved final manuscript.