Functional Characterization of Recombinant Raw Starch Degrading α-Amylase from Roseateles terrae HL11 and Its Application on Cassava Pulp Saccharification

Abstract

Exploring new raw starch-hydrolyzing -amylases and understanding their biochemical characteristics are important for the utilization of starch-rich materials in bio-industry. In this work, the biochemical characteristics of a novel raw starch-degrading -amylase (HL11 Amy) from Roseateles terrae HL11 was firstly reported. Evolutionary analysis revealed that HL11Amy was classified into glycoside hydrolase family 13 subfamily 32 (GH13_32). It contains four protein domains consisting of domain A, domain B, domain C and carbohydrate-binding module 20 (CMB20). The enzyme optimally worked at 50 °C, pH 4.0 with a specific activity of 6270 U/mg protein and 1030 raw starch-degrading (RSD) U/mg protein against soluble starch. Remarkably, HL11Amy exhibited activity toward both raw and gelatinized forms of various substrates, with the highest catalytic efficiency (kcat/Km) on starch from rice, followed by potato and cassava, respectively. HL11Amy effectively hydrolyzed cassava pulp (CP) hydrolysis, with a reducing sugar yield of 736 and 183 mg/g starch from gelatinized and raw CP, equivalent to 72% and 18% conversion based on starch content in the substrate, respectively. These demonstrated that HL11Amy represents a promising raw starch-degrading enzyme with potential applications in starch modification and cassava pulp saccharification.

In-Depth Characterization of Debranching Type I Pullulanase from Priestia koreensis HL12 as Potential Biocatalyst for Starch Saccharification and Modification

“…In this work, HL12Pul exhibited high efficiency in RSIII production (70% recovery) from gelatinized cassava starch. Typically, cassava starch contains 17-20% amylose (a linear chain molecule) and 80-83% amylopectin (a branch polymer of glucose) with high molecular weights [1,45,46]. The different enzyme dosages in starch treatment significantly differed modified starch architecture.…”

“…E. coli DH5α was used as a plasmid propagation host, and E. coli BL21(DE3) (Novagen, Darmstadt, Germany) was used for recombinant protein expression. Recombinant raw starch degrading α-amylase was produced in our laboratory [46]. All chemicals and reagents used were analytical grade and derived from major chemical suppliers (Sigma, Merck, and Fluka).…”

“…To develop an efficient starch hydrolyzing process, the synergistic effects of HL12Pul in starch saccharification were investigated by mixing with recombinant raw starch degrading α-amylase from Roseateles terrae HL11 (HL11Amy) [46], In the mixture design, the HL11Amy was fixed enzyme loading as 1.6 µg protein, while HL12Pul was varied as 0.5, 1.0, and 1.6 µg protein for raw cassava starch hydrolysis. The hydrolysis reaction contained 1% (w/v) raw cassava starch in 50 mM sodium phosphate buffer, pH 6.0, with the enzyme cocktail.…”

Bioengineered Enzymes and Precision Fermentation in the Food Industry

“…The yield and the purity of plant proteins are also closely related to the enzymes used during the extraction. In starch biorefinery, where engineered enzymes such as xylanase, amylase, and pectinase are used to hydrolyze starch, most of the proteins are released as a result of this process [ 94 , 95 ]. Another critical point is plant protein functionality.…”