Molecular Cloning, Characterization, and Application of a Novel Multifunctional Isoamylase (MIsA) from Myxococcus sp. Strain V11

Abstract

A novel multifunctional isoamylase, MIsA from Myxococcus sp. strain V11, was expressed in Escherichia coli BL21(DE3). Sequence alignment revealed that MIsA is a typical isoamylase that belongs to glycoside hydrolase family 13 (GH 13). MIsA can hydrolyze the α-1,6-branches of amylopectin and pullulan, as well as the α-1,4-glucosidic bond in amylose. Additionally, MIsA demonstrates 4-α-D-glucan transferase activity, enabling the transfer of α-1,4-glucan oligosaccharides between molecules, particularly with linear maltooligosaccharides. The K_m, K_cat, and V_max values of the MIsA for amylopectin were 1.22 mM, 40.42 µmol·min^-1·mg^-1, and 4046.31 mM·min^-1. The yields of amylopectin and amylose hydrolyzed into oligosaccharides were 10.16% and 11.70%, respectively. The hydrolysis efficiencies were 55%, 35%, and 30% for amylopectin, soluble starch, and amylose, respectively. In the composite enzyme hydrolysis of amylose, the yield of maltotetraose increased by 1.81-fold and 2.73-fold compared with that of MIsA and MTHase (MCK8499120) alone, respectively.

Keywords: 4-alpha-glucanotransferase; Myxococcus sp. strain V11; isoamylase; maltotetraose; multifunctional starch debranching enzyme.

Figure 1

Multiple sequence alignment of the starch debranching enzyme MIsA from Myxococcus sp. strain V11 with other starch debranching enzymes from various organisms. (Bold red text represents the starch debranching enzyme MIsA from Myxococcus sp. strain V11).

Figure 2

Comparison of the structures of TreX and MIsA. (a). Overall structure of MIsA; red sticks indicate catalytic amino acid residues. (b). Comparison of the structures of TreX (cyan) and MIsA (green). The red circle represents helix 4, the lids of TreX (yellow) and MIsA (blue).

Figure 3

SDS-PAGE gel of MIsA. (a) Analysis of the expression of MIsA via SDS-PAGE. Lane M: standard protein marker; Lane 1: pET-29a (+) empty crude enzyme mixture; Lane 2: MIsA expression strain crude enzyme mixture; Lane 3: MIsA purified by Ni²⁺-affinity chromatography; Lane 4: MIsA purified by dialysis. (b) MIsA was analyzed by Native-PAGE. Lane 1: MIsA expression strain crude enzyme supernatant; lane 2: zymogram analysis of MIsA visualized by activity staining.

Figure 4

Effects of temperature and pH on the activity and stability of MIsA. (a) Optimal temperature. Activity was measured in 50 mM PBS buffer (pH 6.0) for 10 min. (b) Thermal stability. The activity was measured in 50 mM PBS buffer (pH 6.0) for 10 min after incubation of the enzyme at different temperatures for 12 h. (c) The optimal pH. Assays were carried out at 50 °C for 10 min in buffers of varying pH values (3.0–9.0). (d) pH stability. The activity was measured in 50 mM PBS (pH 6.0) at 50 °C for 10 min after the purified enzyme was incubated with buffers of various pH values at 4 °C for 12 h.

Figure 5

Enzyme kinetics of MIsA. (a) Amylopectin as the substrate. (b) Amylose as the substrate.

Figure 6

SEM analysis of insoluble starch granules. (a) Nondebranched starch; (b) starch debranched for 4 h; (c) starch debranched for 9 h. Pictures are shown at low (1 mm) and high (0.01 mm) magnification.

Figure 7

Chain length distribution (CLD) of amylopectin. (a) CLD of amylopectin after treatment with Promozyme^® D2 for 24 h. (b) CLD of amylopectin after treatment with MIsA for 0.5 h. (c) CLD of amylopectin after treatment with MIsA for 1 h.

Figure 8

Determination of glycosidic bond ratios. The number of α-1,6 glycosidic bonds in the control group accounted for 7.24% of the total glycosidic bonds. After 10 min, 30 min, and 60 min of treatment with MIsA, the number of α-1,6 glycosidic bonds are 6.16%, 5.60%, and 5.47%, respectively.

Figure 9

TLC analysis of MIsA activity toward various substrates. MIsA was reacted in 50 mM sodium phosphate buffer (pH 6.0) at 50 °C for 9 h with each substrate. G4, maltotetraose; G5, maltopentaose; G6, maltohexarose; β–CD, β–cyclodextrin; Pul, pullulan; AP, amylopectin; As, amylose; Std, standard of glucose and maltooligosaccharides (G2–G6); reactions on substrates with (+) and without (–) enzymes. The developing solvent was n-butanol/methanol/water at a ratio of 4:2:1 (v/v/v).

Figure 10

The oligosaccharide yields of different substrates treated with MIsA and MTHase. (a) Amylose; (b) amylopectin; (c) soluble starch.