Abstract
Sucrose isomerase (SI), catalysis sucrose to isomaltulose, has been
wildly used in industrial production of isomaltulose. Here, rational
design of Pantoea dispersa SI for improving its thermostability
by predicting and substituting the unstable amino acid residues was
studied using the computational-aided predictor FoldX. Through the
mutation pool, two mutants of SI (V280L, S499F) displayed favorable
characteristics on thermostability. The double mutant V280L/S499F were
further constructed and showed prolonged half-life at 45 ℃, about 9-fold
compared to the wild-type. Accordingly, the melting temperature of
mutant V280L/S499F was improved to 54.2 ℃. To determine the recyclable
ability of mutant V280L/S499F to bioconversion of isomaltulose,
recombinant Corynebacterium
glutamicum/ pXMJ19/pdsi V280L/S499F was
constructed and repeated batch conversion was performed in a 5 L
bioreactor. The results shown that the maximum yield of isomaltulose by
batch conversion reached to 451 g/L with a productivity of 45.1 g/L/h,
and the conversion rate remained 83.2 ± 2.1% even after 15 repeated
batches of biocatalysis. Structure-based molecule molding revealed that
the interiors of mutant V280L/S499F was more tightly packed in ɑ-9 fold
and a new hydrophobic network was formed in ɑ-17, which combined
contributed to improving its thermostability. This work provides new
reference for the sustainable production of isomaltulose.
Introduction
Isomaltulose, a sucrose isomer, is a reducing disaccharide that exists
in natural molasses in a small amount and has a sweetness of 45%
sucrose but is nontoxic and non-cariogenic.[1]Therefore, isomaltulose is an ideal sucrose substitute and is the
healthy sugar certified by United States Food Drug Administration, its
addition and consumption are not restricted.[2] In
addition, isomaltulose has many beneficial healthcare functions and
physiological properties, including inhibiting elevated blood sugar
levels,[3] inhibiting fat
accumulation,[4] improving anti-fatigue
ability,[5] and maintaining the intestinal
microecological balance.[6] However, the process
of chemically synthesizing isomaltose produces by-products and chemical
waste, which increases the cost of product separation and wastewater
treatment.[7] Therefore, the preparation of
isomaltulose by biotransformation technology has been widely
investigated in recent years.
Sucrose isomerase (SI, EC 5.4.99.11), also be known as isomaltulose
synthase, is responsible for conversing sucrose into isomaltulose or
trehalulose along with glucose and fructose.[8]Current investigations of SI are mainly in the mining of novel genes and
property characterizations of SIs. Those currently-reported SIs showed
limited thermostability during the biocatalysis process, such as the SI
of Klebsiella sp. LX3, which has a 1.8 min half-life at 50 °C,[9] SI of Klebsiella pneumonia lost its
40% relative activity after incubating at 50 °C for 20
min,[10] and SI of Erwinia sp . was
completely inactive after incubating at 30°C for 24
h.[11, 12] Although most studies have used enzyme
immobilization or cell surface display technology to improve the
robustness of SI, their industrial applications are still subject to
unsatisfactory thermostability, and the conversion rate often decline
rapidly after several continuous rounds of biocatalysis. Therefore,
modification at the molecular level to improve the thermostability of SI
should be further investigated.
Protein engineering has proven to be an effective approach to enhance
thermostability of enzymes. Protein engineering is subdivided into
directed evolution (irrational design), semi-rational design and
rational design.[13] Although irrational design
and semi-rational design were powerful in enzyme modification at
elevated temperature,[14, 15] they are
time-consuming and laborious. In contrast, rational design based on
computer-aided techniques has greatly accelerated the speed and success
rate of scientific research.[16] Fold X, one of
the most reliable computational design predictors, has been developed to
predict beneficial substitutions related to thermal stability by
performing a rapid evaluation of the Gibbs free energy difference (ΔΔG)[17, 18]. Recently, FoldX has been used to improve
the thermostability of many enzymes by rational design. Luo et
al . obtained a best variant PoOPHM9 with a
thermostability (T5015) of 67.6℃ by
hierarchical iteration mutagenesis using
FoldX.[19] Bi et al . engineered
thermophilic pullulanase by rational design using FoldX predictor, theTm of mutant G692M increased by 3.8 °C,and
half-life is 2.1-fold longer than the wild-type at 70
°C.[20] Wang et al . constructed a quadruple
mutation (S142A/D217V/Q239F/S250Y) variant based on FoldX algorithm, the
half-life of combination mutant increased 41.7-fold at 60
°C.[21] Thus, in-silico energy calculations
(FoldX) may provide a clear guide for the molecular engineering of SI.
Compare with free-enzyme catalysis, while whole-cell biotransformation
provides unique advantages, such as lower-cost preparation, easier
separation of products and simpler recycle course. However, endotoxin or
toxic cell wall pyrogens of non-food-grade host would be an obstacle to
green synthesis of isomaltulose. To solve the potential safety hazards,
some researchers have introduced
SIase genes into non-pathogenic
hosts, including Lactococcus lactisMG1363,[22] B. subtilisWB800,[23]Saccharomyces
cerevisiae, [24] Yarrowia lipolyticaS47.[25] However, recombinant L. lactisMG1363 exhibited low expression level of sucrose isomerase (100 μg/mL),
and Saccharomyces cerevisiae and Yarrowia lipolytica grew
slowly (48-96h). Corynbacterinum glutamicum ATCC13032 (C.
glutamium 13032) is listed as a “generally recognized as safe”
microorganism and has been successfully used as a host for producing
food compounds efficiently, like amino acids, vitamins and organic acids
and rare sugars.[26] Furthermore, C.
glutamicum has many advantages and is superior to other food-grade
strains, such as non-pathogenic, non-codon bias and short fermentation
period. Thus, isomaltulose production by whole-cell biotransformation
using an ideal food-grade host was expected to be more suitable in the
field of food and fermentation research.
Herein, in order to obtain robust SI from a small mutation library via
rational design, computational design software (FoldX5) coupled with
conservation analysis and
functional
region assessment were employed to predict potential stabilizing point
mutations. Then, the best variant was intracellular overexpressed in the
food-grade strain C. glutamium 13032, and the recombinant cells
was further used as whole-cell biocatalyst for the recyclable synthesis
of isomaltulose under the optimized conditions. Finally, Differential
Scanning Fluorimetry (DSF) was used to evaluate the changes in the
thermostability, and molecular dynamic simulation (MD) was used to
elucidate the mechanism for the improved stability. Taken together, this
study provides a new strategy for enhancing the stability of sucrose
isomerase to improve its performance in industrial applications.
Materials and methods