Characterization of a pH and detergent-tolerant, cold-adapted type I pullulanase from Exiguobacterium sp. SH3

Abstract

A pullulanase-encoding gene from psychrotrophic Exiguobacterium sp. SH3 was cloned and expressed in both E. coli and Bacillus subtilis. The functional recombinant enzyme (Pul-SH3) was purified as a His-tagged protein. Pul-SH3 was characterized to be a cold-adapted type I pullulanase with maximum activity at 45 °C. Using fluorescence spectroscopy, the melting temperature of Pul-SH3 was determined to be about 52 °C. The enzyme was able to hydrolyze pullulan, soluble starch, potato starch, and rice flour. It was exceptionally tolerant in the pH range of 4-11, exhibiting maximum activity at pH 8.5 and more than 60% of the activity in the pH range of 5-11. Being a detergent-tolerant pullulanase, Pul-SH3 retained 99, 89, and 54% of its activity at 10% concentration of Triton-X100, Tween 20, and SDS, respectively. The enzyme also exhibited an activity of 80.4 and 93.7% in the presence of two commercial detergents, Rika (7.5% v/v) and Fadisheh (2.5% w/v), respectively. The enzyme was even able to remain active by 54.5 and 85% after 10-day holding with the commercial detergents. Thermal stability of the enzyme could w on silica. Pul-SH3 with several industrially important characteristics seems desirable for cold hydrolysis of starch.

Impact of drinking water treatment and distribution on the microbiome continuum: an ecological disturbance's perspective

“…As no studies have characterized microbial communities in softening Drinking water microbiome continuum 3167 processes, this is the first observation on the dominance of Exiguobacterium-related populations. Exiguobacterium is known to be present in a wide range of pH (4-11), temperature (from permafrost, glacial ice, to hot springs), and salinity (Vishnivetskaya et al, 2009;Rajaei et al, 2015). However, it remains unclear what mechanisms enabled their adaptation to extreme environments.…”

Improving the Stability of Cold-Adapted Enzymes by Immobilization

“…When compared with investigations of the immobilization of mesophilic or thermophilic enzymes, few studies of the immobilization of cold-adapted enzymes have been conducted. Several studies have reported the thermal and storage stabilities of immobilized cold-adapted enzymes along with gene cloning and biochemical characterization [61][62][63][64][65][66]. Although temperature and organic solvents are two major considerations for industrial applications of enzymes, immobilization of cold-adapted enzymes has mainly been evaluated with regard to thermal stability.…”

“…The enzyme leaching shown in adsorption is significantly reduced in covalent binding during reaction and washing [49]. Cold-adapted enzymes have been covalently immobilized on several solid supports, including agarose [63], chitosan [73], Sepharose [62], silica [66], and graphene oxide [72]. The conformational changes resulting from covalent modifications generally lead to reduced enzymatic activity.…”

“…A psychrophilic pullulanase (Exiguobacterium sp.) was shown to lose its activity from 50 • C with almost no activity at 60 • C [66]. Pullulanase immobilized on epoxy-functionalized silica maintained its thermal stability at 50 • C and showed significantly improved thermal stability at 60 • C and 70 • C during 90 min of incubation [66].…”

“…was shown to lose its activity from 50 • C with almost no activity at 60 • C [66]. Pullulanase immobilized on epoxy-functionalized silica maintained its thermal stability at 50 • C and showed significantly improved thermal stability at 60 • C and 70 • C during 90 min of incubation [66]. Similarly, a cold-adapted P. mandelii esterase EstK covalently tethered to graphene oxide exhibited higher thermal stability than that of free enzyme at 20-40 • C, but immobilization did not help improve thermal stability beyond the temperatures [72].…”