Biochemical characterization of a novel thermophilic α-galactosidase from Talaromyces leycettanus JCM12802 with significant transglycosylation activity

Abstract

Thermophilic α-galactosidases have great potentials in biotechnological and medicinal applications due to their high-temperature activity and specific stability. In this study, a novel α-galactosidase gene of glycoside hydrolase family 27 (aga27A) was cloned from Talaromyces leycettanus JCM12802 and successfully expressed in Pichia pastoris GS115. Purified recombinant Aga27A (rAga27A) was thermophilic and thermotolerant, exhibiting the maximum activity at 70°C and retaining stability at 65°C. Like most fungal α-galactosidases, rAga27A had an acidic pH optimum (pH 4.0) but retained stability over a boarder pH range (pH 3.0-11.0) at 70°C. Moreover, the enzyme exhibited strong resistance to most metal ions and chemicals tested (except for Ag(+) and SDS) and great tolerance to galactose (19 mM). The preferable transglycosylation capacity of rAga27A with various substrates further widens its application spectrum. Thus rAga27A with excellent enzymatic properties will be ideal for applications in various industries, especially for the synthesis of galactooligosaccharides.

Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge–charge interactions

“…The thermophilic Talaromyces spp. are known to be potential industrial enzyme producers, as they have been reported to secrete various kinds of hydrolytic enzymes such as mannanase [ 19 ] and α-galactosidase [ 20 ]. However, no β-glucanase of GH16 has been reported from this genus yet.…”

Characterization of a highly stable α-galactosidase from thermophilic Rasamsonia emersonii heterologously expressed in a modified Pichia pastoris expression system

“…Notably, ReGal2 exhibited a significant tolerance to SDS and β-mercaptoethanol with hardly any activity lost when treated with SDS or β-mercaptoethanol up to 200 mM, which is in agreement with the observation that it retained the active oligomeric state during electrophoresis in the presence of these two denaturants. This is quite unique among the so far characterized α-galactosidases, which markedly lost activities in the presence of SDS (Table 5) [9, 10, 12, 31–33]. Even more surprisingly, ReGal2 did not show reduced activity in the presence of urea (up to 200 mM), and maintained approximately up to 80% of its initial activity in the presence of GdnHCl (200 mM), which is a stronger denaturant than urea.…”

“…High structural stability is also an important and desirable feature of enzymes to resist toxic inhibitors either derived from the substrate or generated during the reaction. Compared to extensive studies on the mesophilic α-galactosidases, only a few thermophilic/thermostolerant α-galactosidases have been characterized, most of which are from thermophilic fungi (see a summary in Table 1), e.g ., Neosarotrya fischeri [9, 10], Thermomyces lanuginosus [11], Talaromyces leycettanus [12], and Talaromyces emersonii [13]. These α-galactosidases exhibit various degrees of thermo-activity and thermo-stability as well as resistance to a number of metal ions and some other chemicals (see a summary in Table 5).…”

Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants

“…Thus, when melibiose was used as the substrate, the enzyme synthesized the (1→6)-α-linked bigalactosides ( Figure S2c ), similar to all known melibiases of the GH 36 family [ 16 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ] and to their closely-related GH 27 representatives [ 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ]. Furthermore, α-PsGal formed the (1→4)-α-linked bigalactosides as described for mesophilic terrestrial α- d -galactosidases from Bifidobacterium breve 203 [ 35 ], Lactobacillus reuteri [ 16 ], and the acidic GH 27 family α- d -galactosidases (AgaBf3S) from the bacterium Bacteroides fragilis .…”