Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation

Abstract

Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc.

Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica

“…In contrast, expression in P. pastoris may lead to glycosylation which can have positive effects such as increased stability, as previously shown for a Thermobifida xylanase (Zhao et al, 2015), human aquaporin 10 (Öberg et al, 2011) and Rhizopus chinensis lipase (Yang et al, 2015). Shirke et al also recently reported that glycosylation stabilizes a P. pastoris -expressed cutinase from Thiellavia terrestris by inhibiting its thermal aggregation (Shirke et al, 2017). On the other hand, glycosylation may also have negative effects, as shown for example for a lipase from Rhizomucor miehei which had decreased activity upon N-glycosylation (Liu et al, 2014).…”

Non-covalent conjugation of cutinase from Fusarium sp. ICT SAC1 with pectin for enhanced stability: Process minutiae, kinetics, thermodynamics and structural study

“…Such inter conversion of helices to turns are possibly due to insertion of polysaccharide into helix [14]. The changes in the secondary structure might horseradish peroxidase with starch [17], sodium carboxymethyl cellulose, methyl cellulose, and sodium carboxymethyl chitosan with β-D-glucuronidase [15] and alcohol dehydrogenase with gum Arabic complex [14], and Aspergillus oryzae cutinase with trehalose [11].…”

“…Both the effects plays a crucial role in stabilizing the enzyme molecule by maintaining its native structural integrity at adverse conditions and also inhibits enzyme aggregation [14][15]17]. The enhanced thermostability for wild type Aspergillus oryzae cutinase (wt-AoC) [11], and Fusarium solani cutinase [6] is primarily due to excluded volume effects of trehalose.…”

“…Such approach augments enzymes stability (pH, thermal, kinetic) at both storage and reaction conditions by providing protective shell in the surrounding vicinity. It also aids in hydration by buildup of a more stable water layer around the enzyme surface, and reinforces enzyme rigidity through hydrophobic interactions among the non-polar amino acid residues [11,[15][16][17].…”

“…long chain triglycerides like tricaprylin and triolein [7,8]. It possesses flexible biocatalytic activity on numerous substrates and hence finds budding applications in dairy industry (milk fat hydrolysis), oils and oleo chemical industry (synthesis of active ingredients of daily needed detergents, surfactants, and structure triglycerides), cosmetics industry (synthesis of personal care products ingredients), pharmaceutical, food and agricultural sector (separation of chiral enantiomers, flavor ester synthesis) [9][10][11]. Recently, process intensification for enantioselectivity towards (R,S)-1-phenylethanol under microwave irradiation using cutinase from novel isolate Fusarium sp.…”