Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host

Abstract

Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak activity (81 AU/L) was reached after only 29 h of culturing in 5-L bioreactors. Through simple purification procedure of ammonium sulfate precipitation and affinity chromatography, it was possible to purify the enzyme to apparent homogeneity (25-fold purification factor, at 5 % yield). The optimal conditions for the α-amylase activity were pH 5.0 and a temperature of 40 °C. The α-amylase studied here did not show any obligate requirement for Ca2+ ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice.

A digestive tract expressing α‐amylase influences the adult lifespan of Pteromalus puparum revealed through RNAi and rescue analyses

“…α‐Amylases (α‐1,4‐glucan‐4‐glucanohydrolases, EC 3.2.1.1) belong to family 13 of the glycoside hydrolase group (GH‐13) of the carbohydrate active enzymes (CAZy) system . They are widely used in the food and textile industries . The first insect α‐amylase studied in some detail was reported from the pulse beetle, Callosobruchus chinensis , in which the enzyme was purified and its properties were reported .…”

Metabolic Engineering for Expanding the Substrate Range of Yarrowia lipolytica

“…Important enzymes that release monosaccharides from various substrates have been successfully expressed in this yeast in studies focused on enzyme characterization [60][61][62]. However, no research has been conducted on the ability of these enzymes to allow yeast growth on such substrates when they are the sole carbon source.…”

Filamentous fungi-like secretory pathway strayed in a yeast system: peculiarities of Yarrowia lipolytica secretory pathway underlying its extraordinary performance

“…Enhanced production of the targeted, secretory protein may be achieved by several different strategies, including (i) engineering the DNA coding sequence itself, (ii) modification of a genetic construct structure, (iii) optimization of culturing conditions, and (iv) engineering molecular mechanisms of protein synthesis and secretion pathway (Graf et al 2009; Liu et al 2012). Within each of the proposed strategies, various solutions have been proposed, and some of them were conducted in Y. lipolytica , like (i) optimization of codon usage within the coding sequence (Celińska et al 2015; Celińska 2015; Dulermo et al 2017), (ii) amplification of the gene copy number, manipulation with regulatory elements contained in the genetic construction, mainly promoter (Le Dall et al 1994; Gasmi et al 2011b; Gasmi et al 2011a; Gasmi et al 2012; Dulermo et al 2017; Larroude et al 2018), (iii) bioprocess engineering, optimization of cultivation conditions (Chang et al 1998; Kim et al 2000; Nicaud et al 2002; Celińska et al 2017a), and (iv) co-synthesis of chaperones, manipulation with protein folding, and maturation mechanisms, engineering secretory tags (De Pourcq et al 2012; Celińska et al 2018). Nevertheless, the vast majority of studies on enhanced production of secretory proteins in Y. lipolytica was focused on strategies from (i) to (iii), while the approaches directing secretory pathway were largely neglected.…”