Novel Inhibitory Function of the Rhizomucor miehei Lipase Propeptide and Three-Dimensional Structures of Its Complexes with the Enzyme.

Many proteins are synthesized as precursors, with propeptides playing a variety of roles such as assisting in folding or preventing them from being active within the cell. While the precise role of the propeptide in fungal lipases is not completely understood, it was previously reported that mutations in the propeptide region of the Rhizomucor miehei lipase have an influence on the activity of the mature enzyme, stressing the importance of the amino acid composition of this region. We here report two structures of this enzyme in complex with its propeptide, which suggests that the latter plays a role in the correct maturation of the enzyme. Most importantly, we demonstrate that the propeptide shows inhibition of lipase activity in standard lipase assays and propose that an important role of the propeptide is to ensure that the enzyme is not active during its expression pathway in the original host.

Purification and characterization of a peroxidase isozyme from Indian turnip roots.

A peroxidase isozyme (TP I) from Indian turnip roots ( Brassica rapa ) was purified. TP I had a minimum molecular mass of 45 000 Da as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A far-UV circular dichroism (CD) spectroscopy study of TP I revealed the presence of 44% alpha-helix, 16% beta-sheet, and 40% random structure. The N-terminal sequence of TP I was found to be Gln-Phe-Val-Ile-Pro-Thr-Tyr-Ala-Trp-Gln. Pyromellitic dianhydride (PMDA)-modified TP I showed enhanced thermal stability and p-chlorophenol removal efficiency. In the absence of polyethylene glycol (PEG), PMDA-modified TP I (dose of 50 units mL(-1)) converted 100% p-chlorophenol, while at the same time, native TP I could convert only 85%. In the presence of PEG, PMDA-modified TP I (dose of 0.05 units mL(-1)) converted p-chlorophenol completely in 45 min, while native TP I required 60 min for complete conversion. The K(M) value toward the substrates p-chlorophenol and o-cresol decreased after PMDA modification of TP I, which indicated increased affinity for these substrates.

Purification and properties of the alkaline lipase from Burkholderia cepacia A.T.C.C. 25609.

A Burkholderia cepacia (bacteria) strain, A.T.C.C. 25609, which had been isolated from the bronchial washings of a cystic fibrosis patient, was used to produce lipase. The presence of sodium alginate at an optimal concentration of 8 mg.ml(-1) in the growth medium nearly doubled the production of extracellular lipase activity. The enzyme could be purified with 38-fold purification and 96% activity recovery using a two-step purification protocol. The molecular mass of the purified lipase determined by SDS/PAGE was shown to be 28 kDa. The pH optimum of the purified enzyme was 9 and it was stable up to 12 h at pH 9 and 10. The enzyme has a temperature optimum of 40 degrees C and its half-life (t(1/2)) values were 54 and 46 min at 50 and 60 degrees C respectively. The lipase was found to be stable in the presence of the detergents Tween 20 and Triton X-100. The secondary-structure analysis of lipase by CD spectroscopy showed 52% alpha-helix, 7.7% beta-sheet, 12.6% beta-turn and 27.8% random structure. The lipase was cloned and overexpressed in Escherichia coli. The gene sequence of the cloned lipase was determined and compared with other lipases.

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities.

BACKGROUND: The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. RESULTS: Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The V(max)/K(m) values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50 degrees C, 60 degrees C and 70 degrees C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. CONCLUSION: A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.

Treatment of phenolic wastewater by horseradish peroxidase immobilized by bioaffinity layering.

Horseradish peroxidase was immobilized by bioaffinity layering and used for the treatment of wastewater containing p-chlorophenol. For this purpose, lectin Concanavalin A was bound to Sephadex beads. The glycoenzyme peroxidase was layered upon this Con A layer. Subsequently, alternate layers of the enzyme and Con A were applied. The most efficient design consisted of three layers of Con A and peroxidase each. This immobilized enzyme preparation retained 80% of the activity of the free peroxidase used for immobilization. PEG at the concentration of 0.1 mg ml(-1) was found to prevent enzyme inactivation by the products, although it increased the process time. Thus 60 U ml(-1) of enzyme completely converted the p-chlorophenol (into products) in 4 min in the absence of PEG. On the other hand, only 0.05 U ml(-1) of enzyme was required for this purpose in the presence of PEG but the process required 60 min. Peroxidase converts phenol molecules into free radicals. These free radicals then polymerize and get precipitated. As a further means of minimizing exposure of the enzyme to free radicals and enhancing the reusability, it was decided to remove the enzyme from reaction medium after 10 min. With this strategy, the bioaffinity layered peroxidase preparation could be reused five times without any loss of activity.