Kalyani Mondal

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg−1 h−1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.

Separation of enzymes by sequential macroaffinity ligand-facilitated three-phase partitioning

Pectinase and cellulase were separated from a commercial enzyme preparation called Pectinex Ultra SP-L. This was carried out using a process called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this method, a water-soluble polymer is floated as an interfacial precipitate by adding ammonium sulfate and tert.-butanol. The polymer (appropriately chosen) in the presence of an enzyme for which it shows affinity, selectively binds to the enzyme and floats as a polymer-enzyme complex. In the first step, pectinase was purified (with alginate as the polymer) 13-fold with 96% activity recovery. In the second MLFTPP step, using chitosan, cellulase was purified 16-fold with 92% activity recovery. Both preparations showed a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis. This illustrative example shows that the strategy of sequential MLFTPP can be used to separate important biological activities from a crude broth.

Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate.

Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.

Accelerating Enzymatic Hydrolysis of Chitin by Microwave Pretreatment

Response surface analysis was used to determine optimum conditions [2% (w/v) chitin, 57.5 °C, 38 min] for microwave irradiation of chitin to improve its enzymatic hydrolysis. Vmax/Km of cabbage chitinase toward untreated and microwave‐irradiated chitin was found to be 21.1 and 31.7 nmol h‐1 mg‐2 mL, respectively. Similar improvement was observed in the case of pectinase in its unusual catalytic activity of chitin degradation. It was found that a greater extent of chitin hydrolysis by chitinase was possible after the substrate chitin was irradiated with microwaves.

Macroaffinity Ligand‐Facilitated Three‐Phase Partitioning (MLFTPP) of α‐Amylases Using a Modified Alginate

The crude extracts of α‐amylases when mixed with alginate, tert‐butyl alcohol, and ammonium sulfate resulted in an interfacial precipitate containing polymer‐bound amylase. The precipitate was dissolved in 1 M maltose to recover α‐amylase activity. The recovery of α‐amylases were 74%, 77%, and 92% in the case of Bacillus amyloliquefaciens, wheat germ, and porcine pancreas, respectively. All purified preparations showed a single band on SDS‐PAGE.

Some studies on characterization of three phase partitioned chitosan

Three phase partitioning (TPP) is generally carried out by adding ammonium sulfate and t-butanol to a solution of a macromolecule. Chitosan could be obtained as an interfacial precipitate with 88% yield by subjecting 0.2% (w/v) chitosan solution to TPP with 45% (w/v) ammonium sulfate, with an equal volume of t-butanol at 40 °C. TPP resulted in structural changes which could be seen in its UV spectra, FT-IR spectra and solubility characteristics. TPP-treated chitosan also showed decreased susceptibility towards hydrolysis by chitinase. Thus, TPP can be used as a useful way of altering the properties of chitosan.

κ-Carrageenan as a carrier in affinity precipitation of yeast alcohol dehydrogenase

κ-Carrageenan is a non-toxic polymer from seaweeds, which becomes reversibly insoluble upon the addition of K+. Its conjugate with the dye, Cibacron Blue 3GA, was used to purify alcohol dehydrogenase from crude yeast extract by affinity precipitation. Response surface methodology was used to optimize conditions for affinity precipitation of the enzyme with the polymer–dye conjugate. Recovery of 58% of the enzyme activity with 13.6-fold purification was obtained.

Emerging options in protein bioseparation

Publisher Summary The chapter describes the way downstream processing scientists are meeting protein separation challenges. There is a distinct trend towards simultaneous purification and immobilization strategies. Leads from affinity precipitation have led to smart biocatalyst design which has inbuilt purification step. High performance biocatalyst designs such as cross-linked enzyme aggregate (CLEA) also do not require highly purified enzymes as starting material. Therefore, first one has had integration of upstream and downstream processes. Now, one has integration of purification and immobilization. Thus, there are enough reasons to believe that need (for efficient separation strategies) would continue to breed novel and innovative technologies.

Applications of alginate in bioseparation of proteins.

Alginate is a polysaccharide that is a block polymer consisting of block units of guluronic acid and mannuronic acid. It shows inherent biological affinity for a variety of enzymes such as pectinase, lipase, phospholipase D, α and β amylases and glucoamylase. Taking advantage of its precipitation with Ca2+ and the above-mentioned property, alginate has been used for purification of these enzymes by affinity precipitation, aqueous two phase separation, macroaffinity ligand facilitated three phase partitioning, immobilized metal affinity chromatography and expanded bed affinity chromatography. Thus, this versatile marine resource has tremendous potential in bioseparation of proteins.

Enhancement of catalytic efficiencies of xylanase, pectinase and cellulase by microwave pretreatment of their substrates

Microwave pre-treatment of polygalacturonic acid, xylan and carboxymethylcellulose was found to improve the catalytic efficiencies of pectinase, xylanase and cellulase by 1.5, 2.3 and 1.6 fold, respectively. The microwave effect was distinguished from a pure thermal effect by irradiating at a constant temperature with the help of a non-contact infrared controller. The temperature and time of pre-treatment and substrate concentration during the pre-treatment were optimized by response surface methodology. Scanning electron microscopy revealed significant morphological changes in the substrate as a result of microwave pre-treatment. The time course of enzymatic hydrolysis in each case showed that the use of microwave pre-treated substrates gave higher catalytic rates. Also, a higher degree of bioconversion was observed in each case when microwave pre-treated substrates were used.