Sunita Teotia

An efficient purification process for sweet potato beta-amylase by affinity precipitation with alginate

beta-amylases are used in production of maltose syrup. It is shown that sweet potato beta-amylase can be purified by affinity precipitation with alginate with 80% activity yield and 44 fold purification. SDS-PAGE of the purified protein showed a single band and a subunit weight of 50 kDa. Preliminary data with soybean and barley enzymes indicate that this may be a general method for purification of beta-amylases.

Reversibly soluble macroaffinity ligand in aqueous two-phase separation of enzymes.

Use of alginate as a free bioligand incorporated in an aqueous two-phase system of polyethylene glycol 6000-salt resulted in considerable purification of wheat germ alpha-amylase and sweet potato beta-amylase from their crude extracts. The elution of the enzyme from the free bioligand was facilitated by exploiting the fact that alginate can be reversibly precipitated in the presence of Ca2+. alpha-Amylase could be purified 42-fold with 92% activity recovery. beta-Amylase on the other hand could be purified 43-fold with 90% recovery. Both purified enzymes showed a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Purification of α-amylases using magnetic alginate beads

Magnetic alginate beads were used to purify α-amylases from porcine pancreas, starchzyme, BAN 240L (a commercial purification from Bacillus subtilis), and wheat germ. The beads bound a significant level of α-amylase activity from porcine pancreas, BAN 240L, and wheat germ. In each case, the enzyme activity could be eluted by using 1.0 M maltose, a known competitive inhibitor of α-amylase. In the case of BAN 240L, 3.6-fold purification with 72% recovery of activity was observed. In the case of wheat germ enzyme, starting from the crude extract, 48-fold purification with 70% activity recovery was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis also indicated considerable purification in the latter case.

Magnetite-alginate beads for purification of some starch degrading enzymes

Starch degrading enzymes, viz., β-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. β-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.

Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

Calcium alginate microbeads (212–425 μm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h−1, respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211‐fold purification was observed.

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.

Bioconversion in an aqueous two-phase system using a smart biocatalyst: casein hydrolysis by a α-chymotrypsin derivative

Abstract Casein hydrolysis was carried out in PEG–dextran two-phase system by using α-chymotrypsin immobilized on a water soluble polymer Eudragit S-100. This biocatalyst along with the substrate casein was predominantly found to partition into the PEG phase. Under these optimized conditions, the product was found to partition (84%) in the dextran phase. Removal of dextran phase at an appropriate time interval and replacing it with fresh dextran phase led to considerable enhancement of casein hydrolysis. The biocatalyst could be separated from the PEG phase by lowering the pH to 3.8 and again dissolved in PEG phase by increasing the pH to 7.6. Thus, this smart biocatalyst could be reused for casein hydrolysis in PEG–dextran two-phase aqueous system.

Casein hydrolysis in PEG-salt two phase system by Eudragit-chymotrypsin bioconjugate.

A bioconjugate of α-chymotrypsin and Eudragit S-100 was used in an aqueous two-phase system (polyethylene glycol/phosphate) for casein hydrolysis. More product was obtained by replacing the lower salt phase with a fresh one during the reaction. The bioconjugate could be reused six times for casein hydrolysis.