Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus

Ah-Reum Park,Seung-Won Jang,Jin-Sook Kim,Bong-Seong Koo,Young-Gyun Park,Hyeon-Cheol Lee

doi:10.1186/s12934-017-0841-3

Abstract

BackgroundLactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β-1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization.ResultsUsing superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used.ConclusionThese results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.

Highlights

Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium
Selection of target residues for modifying binding properties The sequence determined for Caldicellulosiruptor saccharolyticus CE (CsCE) obtained from C. saccharolyticus revealed that this cellobiose 2-epimerase (CE) gene was substantially different from that of other species and contained a 1173-bp ORF encoding 390 amino acids
Epimerization catalyzed by CE is likely to proceed with this pair of histidine residues, H377 and H247, which gives rise to a converting the configuration of C2 atom by abstracting and accepting a proton, according to the structural study for the epimerization mechanism of Rhodothermus marinus CE (RmCE) by Fujiwara et al [20]

Summary

Introduction

A synthetic disaccharide, has received increasing interest due to its role as a prebiotic, proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. A few CE 3D structure were reported, revealing mechanistic details Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization. Multiple cellobiose 2-epimerases (CE: EC 5.1.3.11) were reported for industrial process applications using lactose [1,2,3,4]. CE from Caldicellulosiruptor saccharolyticus (CsCE) was reported as a useful enzyme having higher thermostability than other CEs [3, 12, 13]. Using this CsCE, we screened error-prone PCR-generated enzyme variants to identify those with increased thermostability compared to wild type CsCE [14]. The accelerated reaction at high temperature could have great advantages in producing lactulose, as lactulose is a byproduct produced during epimerization from lactose to epilactose, depending on equilibrium concentrations of lactose and epilactose [13, 15, 16]

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