Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes

Abstract

Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases (EC 3.2.1.41, GH57) from an endogenous Cohnella sp., [Coh00831 (KP335161; 1998 bp) and Coh01133 (KP335160: 3678 bp)] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3). The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2), maltotriose (G3) and maltotetraose (G4). The enzymes hydrolyzed pullulan to maltotriose (G3) only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

“…For wielding the enzymes in industry and to understand the cellular organization, kinetics determination of enzymes is always has a crucial role 2 . As shown in Supplementary Table S4, the K m of the glutaminases from different microorganisms are very different and rSAM K m which was found to be 1.8 mM is lower than that of in many other microorganisms.…”

“…Enzymes derived from microorganisms particularly from extremophiles due to their sustainability are appealing for chemical processes designing for many industrial and medical purposes 1,2 . L-glutaminase (EC.…”

Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 β-Glucanase

“…An advantage of industrial application of enzymes is environmental considerations as opposed to chemical catalysts[ 41 ]. Biochemical characterization of this novel bg16M suggests that this enzyme can be applied in beverage industries and medical sectors because of its high activity and thermal stability, as well as its stability and ability to act in the presence of alkali pH, metal ions, and most detergents.…”

Properties of a neutral, thermally stable and surfactant-tolerant pullulanase from worker termite gut-dwelling Bacillus safensis as potential for industrial applications

“…Pullulan is made up of α-1,4 and α-1,6 glycosidic linkage which offers the structure restriction against desiccation and predation ( Farris et al., 2014 ; Ogbo and Nwozor, 2020 ). Breaking down of pullulan requires specific enzyme, as common amylolytic enzymes such as α-amylase and β-amylase could not hydrolyse the compound due to its complexity ( Zebardast et al., 2017 ). In line for various industrial and commercial applications of pullulan, its degradation and enzymatic conversion is highly required, so that variety of useful products could be obtained.…”

“…Therefore, pullulanase (pullulan α-glucano hydrolase; EC 3.2.1.41) has been highly preferred in industrial processes because of its capability to be involved in saccharification of starch, amylopectin, pullulan and related polysaccharides in combination with other amylolytic enzymes and subsequently yield hydrolysis of α-1, 6 bonds in the polysaccharides with the production of maltose, maltotriose and fructose ( Wu et al., 2015 ; Ma et al., 2015 ; Dakhmouche et al., 2021 ). More so, its usefulness as additives, production of malto syrups, pure glucose and fructose and reduction of dental plaque has drawn more attention of its relevance and requirement ( Zebardast et al., 2017 ; Dakhmouche et al., 2021 ).…”