Purification of microbial b-galactosidase from Kluyveromyces fragilis by bioaffinity partitioning

Abstract

This work investigated the partitioning of β-galactosidase from Kluyveromyces fragilis in aqueous two-phase systems (ATPS) by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-β-Dgalactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme β-galactosidase from Kluyveromyces fragilis was developed. In the first step, a system composed of 6% PEG 4000-APGP and 8% dextran 505 was used, where β-galactosidase was strongly partitioned to the top phase (K = 2,330). In the second step, a system formed of 13% PEG-APGP and 9% phosphate salt was used to revert the value of the partition coefficient of β-galactosidase (K = 2 x 10 -5 ) in order to provide the purification and recovery of 39% of the enzyme in the bottom salt-rich phase.

Study on Isolation and Partial Purification of Lactase (β-galactosidase) Enzyme from Lactobacillus Bacteria Isolated from Yogurt

“…In our study an overall recovery (% yield) of 0.306 and a 1.47 purification fold enrichment of the enzyme were obtained after 3 steps. The final specific activity reached 62.8 U/ mg of protein and compared with the values reported by the previous studies [7,11,26,27]. Table 4.…”

Aqueous two-phase extraction (ATPE): An attractive and economically viable technology for downstream processing of Aspergillus oryzae α-galactosidase

“…ATPS has been successfully used to purify extracellular enzymes, for example, xylanase [19,20], b-xylosidase [21], bglucosidase [22,23], endo-polygalacturonase [24], amyloglucosidase [25], b-galactosidase [26,27]. The literature is very sparse regarding the purification of the industrially important enzyme agalactosidase, and there has been no report of the purification of a-galactosidase of A. oryzae with ATPS.…”

Design Strategies for Integrated β‐Galactosidase Purification Processes

“…The purification factors obtained in these systems were equivalent to those from other authors. Silva et al [30] studied the purification of b-galactosidase from Kluyveromyces lactis by ATPS (PEG-phosphate-dextran) and obtained 3.9 and 83 % for the PF and recovery, respectively, adding NaCl to the system and a biospecific ligand, which could have impaired the scaling-up of the process. Silva and Franco [31] described the purification of β-galactosidase from Kluyveromyces fragilis using a PEG/dextran system and reached a 2.8-fold PF value and 57 % enzyme recovery.…”