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A novel cellobiose 2‐epimerase from anaerobic halophilic Iocasia fonsfrigidae and its ability to convert lactose in fresh goat milk into epilactose

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Journal of The Science of Food and Agriculture
Authors:
Sokhoeun Eat
Sokhoeun Eat
Sokhoeun Eat
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Shinta Wulansari at King Mongkut's University of Technology Thonburi
Shinta Wulansari
Prattana Ketbot at King Mongkut's University of Technology Thonburi
Prattana Ketbot
Rattiya Waeonukul at King Mongkut's University of Technology Thonburi
Rattiya Waeonukul
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Abstract and Figures

BACKGROUND Cellobiose 2‐epimerase (CE) has received great attention due to its potential applications in the food and pharmaceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3‐1 (IfCE) was successfully expressed in Escherichia coli and characterized. RESULTS Unlike other CEs, the purified IfCE shows only epimerization activity toward β‐1,4‐glycosidic linkages of disaccharides, including mannobiose, cellobiose and lactose, but not for monosaccharides, β‐1,4‐glycosidic linkages of trisaccharides and α‐1,4‐glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low‐calorie prebiotic sweetener with medicinal and pharmacological properties, was obtained from 10 mg mL⁻¹ lactose. IfCE was highly active against lactose under NaCl concentrations up to 500 mmol L⁻¹, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues may protect the enzyme from Cl⁻ and Na⁺ ions from the environment, respectively. Under normal conditions, IfCE was able to convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been investigated as a safe enzyme for human allergen. CONCLUSION The results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy products by converting lactose that causes digestive problems in people with lactose intolerance into epilactose. © 2024 Society of Chemical Industry.
Amino acid sequence alignment of IfCE compared with those of other characterized CEs: RaCE (AB301953) from Ruminococcus albus, CsCE (FW496353.1) from Caldicellulosiruptor saccharolyticus, RmCE (BAK61777.1) from Rhodothermus marinus JCM9785 and BfCE (BAH23773.1) from Bacteroides fragilis NCTC 9343. The three catalytic residues are highlighted in black and marked with asterisks. Amino acid residues located within the binding pocket of R. marinus CE binding to the sugar moiety of the non‐reducing end (d‐mannose) of the disaccharide, 4‐O‐β‐d‐glucosyl‐d‐mannose,²⁰ which are not fully conserved among CE members, are represented by grey mark. Red letters represent amino acids with positive charge (arginine and lysine) while green letters represent amino acids with negative charge (aspartic and glutamic acids) on the surface of CE structures. Alignments were constructed with ClusterW program on the EMBL‐EBIserver (https://www.ebi.ac.uk/Tools/msa/clustalo/).
Amino acid sequence alignment of IfCE compared with those of other characterized CEs: RaCE (AB301953) from Ruminococcus albus, CsCE (FW496353.1) from Caldicellulosiruptor saccharolyticus, RmCE (BAK61777.1) from Rhodothermus marinus JCM9785 and BfCE (BAH23773.1) from Bacteroides fragilis NCTC 9343. The three catalytic residues are highlighted in black and marked with asterisks. Amino acid residues located within the binding pocket of R. marinus CE binding to the sugar moiety of the non‐reducing end (d‐mannose) of the disaccharide, 4‐O‐β‐d‐glucosyl‐d‐mannose,²⁰ which are not fully conserved among CE members, are represented by grey mark. Red letters represent amino acids with positive charge (arginine and lysine) while green letters represent amino acids with negative charge (aspartic and glutamic acids) on the surface of CE structures. Alignments were constructed with ClusterW program on the EMBL‐EBIserver (https://www.ebi.ac.uk/Tools/msa/clustalo/).
… 
HPLC analysis profiles of the product catalyzed from lactose by IfCE. The reaction was carried out by incubating purified IfCE (1.0 μmol L⁻¹) with 10.0 mg mL⁻¹ lactose at 30 °C and pH 7.0 (50 mmol L⁻¹ Tris–HCl buffer) for 10 min. (A) Standards, lactose, lactulose and epilactose; (B) lactose with denatured enzyme; (C) IfCE reaction to lactose.
HPLC analysis profiles of the product catalyzed from lactose by IfCE. The reaction was carried out by incubating purified IfCE (1.0 μmol L⁻¹) with 10.0 mg mL⁻¹ lactose at 30 °C and pH 7.0 (50 mmol L⁻¹ Tris–HCl buffer) for 10 min. (A) Standards, lactose, lactulose and epilactose; (B) lactose with denatured enzyme; (C) IfCE reaction to lactose.
… 
HPLC analysis profiles of IfCE (1.0 μmol L⁻¹) on 10.0 mg mL⁻¹ of each substrate at 30 °C and pH 7.0 for 10 min. (A, C, E) Each substrate with denatured enzyme; (B, D, F) IfCE reaction to mannobiose, lactose and cellobiose, respectively.
HPLC analysis profiles of IfCE (1.0 μmol L⁻¹) on 10.0 mg mL⁻¹ of each substrate at 30 °C and pH 7.0 for 10 min. (A, C, E) Each substrate with denatured enzyme; (B, D, F) IfCE reaction to mannobiose, lactose and cellobiose, respectively.
… 
Effect of sodium chloride concentration on IfCE activity. (a) HPLC analysis profiles of reactions containing 0–500 mmol L⁻¹ NaCl. The reaction was carried out by incubating IfCE (1.0 μmol L⁻¹) with lactose (10.0 mg mL⁻¹) at 30 °C and pH 7.0 for 10 min. (A) Standards, lactose and epilactose; (B) reaction with denatured enzyme; (C) reaction without NaCl; (D–F) reaction under 100, 300 and 500 mmol L⁻¹ NaCl, respectively. (b) Changing concentrations of epilactose and lactose after incubation under various concentrations of NaCl. Different letters indicate significant differences within groups at P < 0.05. Values are the means from triplicate experiments ± standard deviations.
Effect of sodium chloride concentration on IfCE activity. (a) HPLC analysis profiles of reactions containing 0–500 mmol L⁻¹ NaCl. The reaction was carried out by incubating IfCE (1.0 μmol L⁻¹) with lactose (10.0 mg mL⁻¹) at 30 °C and pH 7.0 for 10 min. (A) Standards, lactose and epilactose; (B) reaction with denatured enzyme; (C) reaction without NaCl; (D–F) reaction under 100, 300 and 500 mmol L⁻¹ NaCl, respectively. (b) Changing concentrations of epilactose and lactose after incubation under various concentrations of NaCl. Different letters indicate significant differences within groups at P < 0.05. Values are the means from triplicate experiments ± standard deviations.
… 
Conversion of lactose in fresh goat milk into epilactose by IfCE (5.0 μmol L⁻¹). (a) HPLC analysis profiles of the IfCE reaction to fresh goat milk: (A) standard lactose and epilactose; (B) goat milk without enzyme; (C, D) reaction at 30 °C for 10 and 60 min, respectively. (b) Concentrations of lactose and epilactose changed during reaction using incubation times of 10, 20 30, 40, 50 and 60 min of IfCE with fresh goat milk. Different letters indicate significant differences within groups at P < 0.05. Values are the means from triplicate experiments ± standard deviations.
Conversion of lactose in fresh goat milk into epilactose by IfCE (5.0 μmol L⁻¹). (a) HPLC analysis profiles of the IfCE reaction to fresh goat milk: (A) standard lactose and epilactose; (B) goat milk without enzyme; (C, D) reaction at 30 °C for 10 and 60 min, respectively. (b) Concentrations of lactose and epilactose changed during reaction using incubation times of 10, 20 30, 40, 50 and 60 min of IfCE with fresh goat milk. Different letters indicate significant differences within groups at P < 0.05. Values are the means from triplicate experiments ± standard deviations.
… 
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Research Article
Received: 4 January 2024 Revised: 8 June 2024 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.13680
A novel cellobiose 2-epimerase from
anaerobic halophilic Iocasia fonsfrigidae and
its ability to convert lactose in fresh goat milk
into epilactose
Sokhoeun Eat,aShinta Wulansari,aPrattana Ketbot,bRattiya Waeonukul,a,b
Patthra Pason,a,b Ayaka Uke,cAkihiko Kosugi,cKhanok Ratanakhanokchaia,b
and Chakrit Tachaapaikoona,b
*
Abstract
BACKGROUND: Cellobiose 2-epimerase (CE) has received great attention due to its potential applications in the food and phar-
maceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3-1
(IfCE) was successfully expressed in Escherichia coli and characterized.
RESULTS: Unlike other CEs, the purified IfCE shows only epimerization activity toward ⊎-1,4-glycosidic linkages of disaccha-
rides, including mannobiose, cellobiose and lactose, but not for monosaccharides, ⊎-1,4-glycosidic linkages of trisaccharides
and ⊍-1,4-glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against
mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low-calorie prebiotic sweetener
with medicinal and pharmacological properties, was obtained from 10 mg mL
−1
lactose. IfCE was highly active against lactose
under NaCl concentrations up to 500 mmol L
−1
, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic
and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues
may protect the enzyme from Cl
−
and Na
+
ions from the environment, respectively. Under normal conditions, IfCE was able to
convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been
investigated as a safe enzyme for human allergen.
CONCLUSION: The results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy prod-
ucts by converting lactose that causes digestive problems in people with lactose intolerance into epilactose.
© 2024 Society of Chemical Industry.
Supporting information may be found in the online version of this article.
Keywords: cellobiose 2-epimerase; epilactose; goat milk; halophilic enzyme; Iocasia fonsfrigidae
INTRODUCTION
Milk is one of the most suitable food sources for human beings,
containing many nutrients. However, milk is high in lactose (4-O-
⊎-D-galactopyranosyl-D-glucose), a disaccharide composed of
galactose and glucose, occurring naturally in milk, accounting
for approximately 35% of the total solids in normal milk.
1
The
presence of lactose in milk causes lactose intolerance or lactose
malabsorption, which is a global health issue, because the activity
of lactase (⊎-galactosidase) declines in the human small intestine
as age increases.
2
About 65% of the adult digestive system worldwide is unable to
digest lactose, and its prevalence even reaches 95% in Asia. Fer-
mentation of indigested lactose by colonic microbiota in the large
intestine causes gastrointestinal symptoms including abdominal
pain, bloating, gas, nausea and diarrhea. Moreover, the avoidance
of dairy products by lactose-intolerant individuals often leads to
insufficient calcium, zinc and vitamin D consumption and may
cause adverse health outcomes, such as a significant reduction
in bone mineral density and fractures.
2,3
Therefore, the
*Correspondence to: C Tachaapaikoon, Division of Biochemical Technology,
School of Bioresources and Technology, King Mongkut's University of Technol-
ogy Thonburi, Bangkok 10150, Thailand. E-mail: chakrit.tac@kmutt.ac.th
aDivision of Biochemical Technology, School of Bioresources and Technology,
King Mongkut's University of Technology Thonburi, Bangkok, Thailand
bExcellent Center of Enzyme Technology and Microbial Utilization, Pilot Plant
Development and Training Institute, King Mongkut's University of Technology
Thonburi, Bangkok, Thailand
cBiological Resources and Post-harvest Division, Japan International Research
Center for Agricultural Sciences, Tsukuba, Japan
J Sci Food Agric 2024 www.soci.org © 2024 Society of Chemical Industry.
1
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