Improving Thermostability and Catalytic Behavior of l-Rhamnose

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Biotechnology and Biological Transformations

Improving thermostability and catalytic behavior towards Dallulose of L-rhamnose isomerase from Caldicellulosiruptor obsidiansis OB47 by site-directed mutagenesis Ziwei Chen, Jiajun Chen, Wenli Zhang, Tao Zhang, Cuie Guang, and Wanmeng Mu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05107 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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Journal of Agricultural and Food Chemistry

Improving thermostability and catalytic behavior towards D-allulose of L-rhamnose isomerase from Caldicellulosiruptor obsidiansis OB47 by

site-directed mutagenesis

Ziwei Chen, † Jiajun Chen, † Wenli Zhang, † Tao Zhang, † Cuie Guang, † and Wanmeng Mu*, †, § †

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.

§

International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu 214122, China.

*

Corresponding author.

Address: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, P. R. China. Tel: (86) 510-85919161. Fax: (86) 510-85919161. E-mail address: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT

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D-Allose, a rare sugar, is an ideal table sugar substitute and has many advantageous

3

physiological functions. L-Rhamnose isomerase (L-RI) is an important D-allose-producing

4

enzyme but exhibits a comparatively low catalytic activity on D-allulose. In this study, an

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array of hydrophobic residues located within the β1-α1-loop was solely or collectively

6

replaced with polar amino acids by site-directed mutagenesis. A group of mutants were

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designed to weaken the hydrophobic environment and strengthen the catalytic behavior on

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D-allulose. Compared to the wild-type enzyme, the relative activities of V48N/G59N/I63N

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and V48N/G59N/I63N/F335S mutants were increased by 105.6% and 134.1% acting on

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D-allulose, respectively. Another group of mutants were designed to enhance

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thermostability. Finally, the t1/2 values of mutant S81A were increased by 7.7 and 1.1 h at

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70 and 80°C, respectively. These results revealed that site-directed mutagenesis is efficient

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for improving thermostability and catalytic behavior towards D-allulose.

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KEYWORDS: D-allose, L-rhamnose isomerase, catalytic behavior, thermostability, site-

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directed mutagenesis

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INTRODUCTION

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In the natural environment, only seven monosaccharides are common sugars that are

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abundant in nature, such as D-glucose and D-fructose. The majority of monosaccharides

20

are rare sugars, which are defined by the International Society of Rare Sugars (ISRS) as

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monosaccharides and monosaccharides derivatives that barely appear in nature.1 D-Allose,

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an extensively studied rare sugar, has 80% relative sweetness to sucrose but is non caloric

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and nontoxic2, 3. Therefore, D-allose is an ideal table sugar substitute and food additive and

24

is beneficial to weight loss. Also, D-allose displays many salutary physiological functions,

25

such as anti-tumor, anti-cancer,4 cryoprotective,5 neuroprotective,6 anti-osteoporotic,7 anti-

26

inflammatory,8

27

physiological functions and health benefits of D-allose have been reviewed in detail.11 D-

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Allose has huge application potential in the food systems, clinical treatment and health care

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fields because of its remarkable physiological functions. However, chemical synthesis of

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D-allose has many disadvantages, including a low conversion rate, chemical pollution and

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byproduct generation.12 The enzymatic production of D-allose was widely investigated in

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recent years.

anti-hypertensive,9

and

immunosuppressant

functions.10

More

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L-Rhamnose isomerase (L-RI, EC 5.3.1.14), one type of aldose-ketose isomerase,

34

catalyzes the conversion between L-rhamnose and L-rhamnulose. L-RI has a broad

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substrate spectrum and can also catalyze the isomerization of D-allulose and D-allose.13 L-

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RI, as an important D-allose-producing enzyme, has been extensively studied. To date, 3



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from various microorganisms, more than ten L-RIs catalyzing the isomerization reaction

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between D-allulose and D-allose have been cloned and identified, such as Clostridium

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stercorarium ATCC 35414 L-RI,14 Thermobacillus composti KWC4 L-RI,15 Bacillus

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subtilis WB600 L-RI,16 Dictyoglomus turgidum DSMZ 6724 L-RI,17 Bacillus pallidus Y25

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L-RI,18 Thermotoga maritima ATCC 43589 L-RI,19 Caldicellulosiruptor saccharolyticus

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ATCC

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saccharolyticum NTOU1 L-RI,22 and Pseudomonas stutzeri L-RI.13 However, these

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investigations primarily focused on screening strains and property characterizations of L-

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RIs. These characterized L-RIs show ultralow catalytic activity on D-allulose, which limits

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the application of L-RIs in the industrial production of D-allose. Furthermore, few studies

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are available in regards to enhancing the thermostability and catalytic behavior by

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molecular modification, which are two important factors in the enzymatic production of D-

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allose. To date, the crystal structures and the catalytic mechanisms of Escherichia coli L-

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RI,23 Bacillus halodurans ATCC BAA-125 L-RI,24 and P. stutzeri L-RI have been

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resolved.25 In 2010, the effect of the C-terminal region and residue Ser329 of P. stutzeri L-

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RI corresponding to Phe336 in E. coli L-RI on substrate specificity was elaborated by

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Yoshida et al with site-directed mutagenesis.26 To date, only this paper has focused on the

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site-directed mutagenesis of L-RI. However, the variation of specific activity of P. stutzeri

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L-RI on D-allulose and D-allose has not been further explored.

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43494

L-RI,20

Mesorhizobium

loti

L-RI,21

Thermoanaerobacterium

Previously, the wild-type L-RI from Caldicellulosiruptor obsidiansis OB47 was 4


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characterized in our laboratory.27 Although C. obsidiansis OB47 L-RI exhibits the highest

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catalytic activity (19.4 U/mg) on D-allulose compared with other reported L-RIs, such as

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T. composti KWC4 L-RI (1.7 U/mg) and T. maritima ATCC 43589 L-RI (1.1 U/mg),19, 28

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it still cannot remotely meet the needs of industrial production of D-allose. In this work,

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we built a model of C. obsidiansis OB47 L-RI on the basis of the B. halodurans ATCC

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BAA-125 L-RI structure. Therefore, we rationally designed site-directed mutagenesis on

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the grounds of reported structural information of L-RIs to further improve thermostability

64

and catalytic activity on D-allulose of C. obsidiansis OB47 L-RI, which is conducive to

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industrial production of D-allose.

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MATERIALS AND METHODS

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Strains, Reagents and Chemicals. The E. coli DH5α and BL21 (DE3) strains were

69

purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The plasmid harboring the

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C. obsidiansis OB47 L-RI gene was constructed in our previous work27. The reagents used

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for site-directed mutagenesis of wild-type L-RI gene were obtained from Generay Biotech

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Co., Ltd. (Shanghai, China). Isopropyl--D-1-thiogalactopyranoside (IPTG) for induction

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was from Sigma (St. Louis, MO, USA). The Ni2+-chelating affinity chromatography resin

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was provided by GE (Uppsala, Sweden). Electrophoretic reagents were obtained from Bio-

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Rad (Hercules, CA, USA). Other chemicals were from Sinopharm Chemical Reagent

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(Shanghai, China) or Sigma (St. Louis, MO, USA). 5



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Molecular Modeling and Docking. The homology modeling of the three-dimensional

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structure of wild-type enzyme and mutants was conducted by the SWISS-MODEL online

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server (http://www.expasy.ch/swissmod/ SWISS-MODEL.html) using the crystal structure

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of B. halodurans ATCC BAA-125 L-RI (PDB number: 3P14) as a template (sequence

82

identity = 55%).29-31 The model energy minimization was performed using the Discovery

83

Studio package (Accelrys, CA, USA). The accuracy of wild-type enzyme and mutant

84

models was examined by the SAVES server.32, 33 The stereochemical quality was verified

85

by Procheck with its Ramachandran plot module.34 The obtained models were delineated

86

and presented with the Pymol Molecular Graphics Software.

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The L-rhamnose and D-allulose models were constructed by the GlycoBioChem

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PRODRG2 online server (http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg/submit.html).

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The ligand energy minimization was implemented by Chem3D Pro14.0.35 L-Rhamnose and

90

D-allulose were used as ligands. Correspondingly, the wild-type enzyme and mutant

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models were used as acceptors for docking. The docking procedure was executed by the

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Autodock 4.2 software package.36 The obtained models were further handled by a battery

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of programs in AutoDock Tool, such as adding hydrogen atoms, calculating charge and

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removing water molecules.

95 96

Site-Directed Mutagenesis. Site-directed mutagenesis of C. obsidiansis OB47 L-RI was 6


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manipulated by one-step PCR methods using a TaKaRa MutantBEST Kit (TaKaRa, Dalian,

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China). The mutants were divided into two groups: One group, including S81A, S81Q,

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S88R, V421I and I343A, for enhancing the thermostability; and another group, including

100

V48N, G59N, G60T, G62T, I63N, I101N, F335C, F335S, V48N/ G59N/ I63N and V48N/

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G59N/ I63N/F335S, for improving the catalytic activity towards D-allulose. The

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recombinant plasmid containing the wild-type C. obsidiansis OB47 L-RI gene was used as

103

the template. All primers used for mutagenesis are shown in Table S1. After PCR

104

amplification, the obtained products were digested and purified by DpnI. The gene

105

sequences of various mutants were verified by Sangon Biotech Co., Ltd. (Shanghai, China).

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Heterologous Expression and Purification. The mutant plasmids were introduced into E.

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coli DH5α and BL21 (DE3) for gene cloning and enzyme overexpression, respectively.

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The recombinant BL21 strains were cultivated in Luria-Bertani medium (LB, 10 g L-1

110

tryptone, 10 g L-1 NaCl, and 5 g L-1 yeast extract) with ampicillin (100 µg/mL) at 37°C and

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200 rpm. The IPTG was added to the LB medium at a final concentration of 1 mM until

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the OD600 attained 0.5-0.7. The recombinant cells were induced for 6 h at 28°C. After that,

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the induced recombinant cells were harvested by centrifugation at 8000 g for 10 min. Then,

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the collected cells were washed twice using distilled water and stored at - 20°C.

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The purification procedures for the wild-type enzyme and mutants were accomplished

116

in the cold room. The pelleted cells were suspended in cell lysis buffer (pH 8.0) and 7



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disrupted by sonication for 16 min (on 1 s, off 2 s) with a Scientz-II D ultrasonic

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homogenizer (Scientz Biotechnology, Ningbo, China). The cellular lysates were

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centrifuged at 10000 g for 15 min to remove the cell fragments, and then the supernatant

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was collected and filtered through a 0.45 μm water phase filter. Fast Protein Liquid

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Chromatography (FPLC, ÄKTA Purifier System, GE Healthcare) was used for purification

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of the wild-type enzyme and mutants. The column was washed using 5 column volume

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(CV) ultrapure water at 1 mL/min flow rate. The filtrate was loaded on a Ni2+-chelating

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Sepharose Fast Flow resin column (8.9× 64 mm, GE Healthcare). The column was pre-

125

equilibrated with 12 CV fresh binding buffer (50 mM Tris-HCl, 500 mM NaCl, pH 8.0) at

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a 0.6 mL/min flow rate. Afterwards, the unbound and unwanted enzymes were eliminated

127

from the resin column using 6 CV washing buffer (50 mM Tris-HCl, 500 mM NaCl, and

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50 mM imidazole, pH 8.0) at 1 mL/min flow rate. Lastly, the target proteins were eluted

129

using 6 CV elution buffer (50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0)

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at 1 mL/min. The fractions displaying catalytic activity were pooled and dialyzed against

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50 mM Tris-HCl buffer (pH 8.0) containing EDTA for 12 h to remove metal ions.

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Subsequently, the protein was dialyzed against Tris-HCl buffer (pH 8.0) to remove EDTA.

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The protein concentrations were measured by the Lowry method using bovine serum

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albumin as the reference.37 The purity and molecular weights of wild-type enzyme and

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mutants were checked using 12% sodium dodecyl sulfate polyacrylamide gel

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electrophoresis (SDS-PAGE) with Coomassie brilliant blue R250 staining. 8


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Isomerization Activity. The catalytic activity of wild-type enzyme and mutants towards

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D-allulose was determined by assaying the formation of D-allose. Under optimal reaction

140

conditions, the enzymatic reactions were implemented at 85°C with 50 mM N-(2-

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Hydroxyethyl) piperazine-N-3-propanesulfonic acid (HEPPS) buffer (pH 8.0) containing

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40 mM D-allulose, 1 mM Co2+ and 0.05 μM purified enzyme. After 10 min, the reaction

143

mixture was boiled for 15 min to stop the enzyme reaction. The catalytic activity of wild-

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type enzyme and mutants towards L-rhamnose was investigated by assaying the

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accumulated amount of L-rhamnulose. The reaction conditions of catalytic activity acting

146

on L-rhamnose are the same as acting on D-allulose.

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The concentration of D-allose was determined by high-performance liquid

148

chromatography (HPLC) with a refractive index (IR) detector (2414, Waters, USA) and a

149

Ca2+ ligand exchange column (6.5 mm × 300 mm, Sugar-Pak 1, Waters Corp., USA). The

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column was eluted with ultrapure water at a column temperature of 85°C with a flow rate

151

of 0.4 mL/min. The concentration of L-rhamnulose was determined using a modified

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cysteine-sulfuric acid-carbazol method.38 First, 500 μL reaction mixture was diluted and

153

was added to 100 μL of 1.5% cysteine hydrochloride solution. After blending, 3 mL of 75%

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sulfuric acid and 100 μL of ethanol-carbazole were added in turn. Then, the generated

155

mixture was immediately incubated at 60°C for 10 min and the absorbance was promptly

156

measured at 540 nm. One unit of enzyme activity was defined as the amount of enzyme 9



157

catalyzing the generation of 1 μmol monosaccharide per minute at 85°C and pH 8.0.

158 159

Mutation for Thermostability. To investigate the thermostability of mutants, the half-life

160

(t1/2) and melting temperature (Tm) were measured. For t1/2 determination, the wild-type

161

enzyme and mutants were pre-incubated at 70 and 80°C. The samples were discontinuously

162

withdrawn at specific times, and the residual activity was later assayed at pH 8.0 and 85°C.

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The initial activity without incubation was set as 100%.

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A differential scanning calorimeter (Nano DSC III, TA Instrument, USA) equipped with

165

a Platinum Capillary Cell was used for Tm value determination. After vacuum

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degasification (635 mmHg), the dialyzed buffers and proteins were loaded into reference

167

and sample cells, respectively. The scanning was carried out at 3 atm air pressure from 25

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to 100°C with a heating rate of 1 °C/min after pre-equilibration for 10 min. The DSC data

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of the wild-type enzyme and mutants were analyzed using the TA Instruments Nano

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Analyze software. The Two State Scaled model was selected in the fitting process after

171

baseline-corrected fitting.

172 173

Production of D-Allose from D-Allulose. The production of D-allose from D-allulose was

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implemented in 5 mL reaction mixtures containing 50 mM HEPPS buffer (pH 8.0), 1 mM

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Co2+ and 5 μM of purified enzyme. Twenty-five g/L D-allulose was used as the initial

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substrate concentration. Considering the thermostability and productivity, the conversation 10


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temperature was set at 60°C. The reaction mixture was inactivated and detected by HPLC

178

at given times to determine the concentration of D-allose. All experiments were

179

implemented in triplicate.

180 181

RESULTS AND DISCUSSION

182

Expression and Purification. After overexpression, the wild-type enzyme and mutants

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were purified. As shown in Figure S1, an approximately 48 kDa band was visible in the

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SDS-PAGE gel, which was in agreement with the theoretical molecular weight. This result

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manifested that the folding of the mutant proteins was correct and that expression and

186

purification are not affected by mutagenesis.

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Structural Modeling. Homology modeling is the most effective method to predict

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unresolved protein structure. Homology modeling is based on two principles: the first point

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is that the protein three-dimensional structure is exclusively determined by the amino acid

191

sequence, and could be theoretically inferred from the primary sequence; and the second

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point is that protein three-dimensional structure is highly conservative in the course of

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protein evolution. The B. halodurans ATCC BAA-125 L-RI shared 55% sequence identity

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with C. obsidiansis OB47 L-RI, and thus, was chosen as the template for homology

195

modeling. The models of wild-type (as shown in Figure 1A) and variant C. obsidiansis

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OB47 L-RI were constructed on the basis of the crystal structure of B. halodurans ATCC 11



197

BAA-125 L-RI (PDB ID: 3P14) using the SWISS-MODEL server. After the energy

198

minimization, the quality of the obtained model was evaluated using the VERIFY-3D

199

procedure from the SAVES server. The results of VERIFY-3D revealed that 94.98% of the

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amino acid residues possessed an average 3D-1D score ≥ 0.2 in 3D-1D structural

201

compatibility, which was considerably greater than the minimal quality requirement (80%).

202

The Ramachandran plot (Figure S2) exhibited that the amino acid residues at the

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percentages of 90.3%, 9.0% and 0.7% were located in the most favored regions,

204

additionally allowed regions and generously allowed regions, respectively. Moreover,

205

amino acid residues were scarcely located in disallowed regions. The monomer structures

206

of C. obsidiansis OB47 L-RI (cyan) and B. halodurans ATCC BAA-125 L-RI (warm pink)

207

were superimposed (Figure 1B) with a 0.120 of root-mean-square deviation (RMSD) value.

208

The result of superimposition indicated that the structures of C. obsidiansis OB47 L-RI and

209

template were very similar. All of these results revealed that the acquired 3D models were

210

applicable and could be used for further structural analysis.

211

The structural arrangement of C. obsidiansis OB47 L-RI is the (β/α)8-barrel

212

conformation (Figure 1), which is composed of alternating connections between eight α-

213

helices (α1-α8) and eight β-strands (β1-β8). Moreover, the C. obsidiansis OB47 L-RI

214

structure has additional α-helical domains (α0, α9, α10, α11, α12) and an extended flexible

215

loop, which is similar to other resolved L-RI structures and may contribute to the

216

association between subunits and the combined action with active sites in the catalytic 12


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center, respectively.23, 25 It is thought that the (β/α)8-barrel conformation of L-RIs is the

218

most widespread and stable fold in characterized and resolved enzymes. Having intrinsic

219

stability, this (β/α)8-barrel structure can serve as the core scaffold for molecular

220

modification aiming at thermostability and catalytic activity. Notably, the flexible loop

221

above the catalytic core, which determines substrate specificity, is the most attractive

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candidate for improving catalytic activity on D-allulose by site-directed mutagenesis

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according to the reported E. coli L-RI structural information.

224 225

Effect of Mutation on Catalytic Behavior. In 2000, Korndörfer et al. reported that in the

226

E. coli L-RI structure, the β1-α1-loop, which is a flexible loop domain consisting of a series

227

of hydrophobic residues (Asp52-Arg78), is probably in charge of the recognition of

228

substrates. This β1-α1-loop of E. coli L-RI is similar to a lid or switch partly covering the

229

catalytic pocket to control the entry of L-rhamnose. Furthermore, this β1-α1-loop together

230

with several non-conservative hydrophobic residues (Ile105, Tyr106 and Phe336) creates

231

a hydrophobic region encompassing the substrate of the C6-methyl group. It revealed that

232

E. coli L-RI prefers the L-rhamnose with a C6-methyl group over the substrates with a C6-

233

oxhydryl group, such as D-allose and D-allulose. Particularly, in E. coli L-RI, V53, I67 and

234

I105 (in C. obsidiansis OB47 L-RI corresponding to V48, I63 and I101) have a

235

hydrophobic stacking interaction and a significant effect on the recognition of substrate.23

236

In 2007, a similar β1-α1-loop (Gly60-Arg76) was found in the P. stutzeri L-RI, which 13



237

exhibits a broad substrate specificity. However, the difference is that the β1-α1-loop of P.

238

stutzeri L-RI covers the adjacent subunit molecule in connection with the substrate binding.

239

In addition, the β1-α1-loop of P. stutzeri L-RI only forms a hydrophobic interaction with

240

the substrate instead of a hydrophobic pocket, which results in a slight recognition for the

241

C6 position. This can explain why P. stutzeri L-RI has a broader substrate specificity than

242

E. coli L-RI.25

243

In E. coli L-RI, Phe336 (in C. obsidiansis OB47 L-RI corresponding to Phe335) in the

244

vicinity of conservative residues has a significant impact on the substrate specificity.23

245

However, this site is a hydrophilic serine in D. turgidum DSMZ 6724 L-RI, M. loti L-RI

246

and P. stutzeri L-RI (S329) and a hydrophilic cysteine in Caldilinea aerophila L-RI (a

247

hypothetical L-RI in GenBank, NCBI number: WP_014435274.1) (Figure S3).

248

Furthermore, to investigate the effect of S329 in P. stutzeri L-RI on substrate specificity,

249

Yoshida et al designed four mutants including S329F, S329K, S329L and S329A. The

250

results showed that the kcat/Km of S329F acting on D-allose was distinctly lower. To

251

summarize, this site together with the “β1-α1-loop” creates the hydrophobic catalytic

252

environment which possibly has an enormous effect on recognition of substrate according

253

to these L-RI structural information which has been verified.

254

The possible location of the β1-α1-loop (Asp47-Agr73) in the structure model of C.

255

obsidiansis OB47 L-RI was determined by sequence alignment and structural analysis. As

256

shown in Figure 2A, the surface model of C. obsidiansis OB47 L-RI β1-α1-loop (raspberry) 14


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above the central tunnel (green) is also similar to a lid and may be involved in the

258

recognition of substrate. A hydrophobic cavity between the β1-α1-loop and the catalytic

259

pocket (yellow) can be clearly observed from lateral view (Figure 2B). The surface models

260

superimposed with the cartoon model are presented in Figure 2C and 2D. To improve the

261

catalytic activity of C. obsidiansis OB47 L-RI on D-allulose (C6-oxhydryl group), which

262

is good for the industrial production of D-allose, a group of mutants was designed by

263

weakening the hydrophobic environment created by the “β1-α1-loop”. In C. obsidiansis

264

OB47 L-RI, a group of hydrophobic residues comprised of four continuous glycines (G59,

265

G60, G61 and G62) may exert a strong hydrophobic interaction. Therefore, five

266

hydrophobic residues (V48, G59, G60, G62 and I63) located within the β1-α1-loop and

267

two subsidiary residues I101 and F335 (in E. coli L-RI corresponding to Ile105 and F336,

268

respectively) were selected as the mutation sites and replaced with hydrophilic residues.

269

Hence, eight single-point mutants (V48N, G59N, G60T, G62T, I63N, I101N, F335C and

270

F335S) and two multiple mutants (V48N/G59N/I63N and V48N/G59N/I63N/F335S) were

271

designed for catalytic behavior.

272

The catalytic activities of wild-type enzyme and mutants towards L-rhamnose and D-

273

allulose were determined at optimal reaction conditions as previously described,27 and the

274

activity of the wild-type enzyme was set as 100%. As shown in Table 1, compared with

275

the wild-type enzyme, the relative activities of V48N, G59N, G62T, I101N, F335C and

276

F335S acting on D-allulose were increased by 68.6%, 61.4%, 36.1%, 36.8%, 87.4% and 15



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31.7%, respectively. Moreover, the relative activities of all mutants acting on L-rhamnose

278

were visibly decreased. In particular, the relative activities of V48N/G59N/I63N and

279

V48N/G59N/I63N/F335S were increased by 105.6% and 134.1% on D-allulose, and

280

decreased by 38.5% and 39.4% on L-rhamnose, respectively. This may suggests that

281

multiple mutation sites exert a synergistic effect. This result is largely consistent with the

282

design principle of mutation. The cartoon models of the wild-type enzyme (A) and mutant

283

V48N/G59N/I63N/F335S (B) are shown in Figure 3. Furthermore, to elucidate vividly the

284

variation of the catalytic pocket, their surface models are presented in Figure 4. After the

285

residues of V48 and F335 were respectively substituted by N48 and S335, their positions

286

were closer to the substrate and the catalytic pocket was partly shrunk. The shrinking of

287

the catalytic pocket enhanced the hydrophilic environment and the interaction with the

288

substrate of the C6-oxhydryl group. After G59 was replaced by N59, the position of the

289

residue shifted to the inside from the edge of the catalytic pocket. However, when the I63

290

was replaced by 63N, the side chain of the 63N residue diverged the central tunnel. This

291

finding could explain why the relative activity of I63N towards D-allulose was not

292

increased.

293 294

Effect of Mutation on Thermostability. To enhance the thermostability of C. obsidiansis

295

OB47 L-RI, the PDB file of the model was uploaded to the Hotspot Wizard 3.0 online

296

server

(https://loschmidt.chemi.muni.cz/hotspotwizard/), 16


which

can

automatically

Page 17 of 36


297

establish mutation libraries and design site-specific mutation for protein stability according

298

to the amino acid frequency and evolutionary information from three large databases.39 The

299

server recommended many sites which may alter the thermostability of L-RI. By the

300

analysis of C. obsidiansis OB47 L-RI structural model, it is observed that two residues S81

301

and S88 located in α1 region may generate interplay with V421 and I343 located in α1 and

302

α8 regions, respectively. Thus, five mutants, S81A, S81Q, S88R, V421I and I343A, were

303

designed for further studies. The t1/2 of the wild type enzyme and mutants was determined

304

at 70 and 80°C. As shown in Table 2, in contrast to the wild-type enzyme, the t1/2 of S88R,

305

V421I and I343A was obviously lower at 70 and 80°C. Interestingly, the t1/2 of S81A was

306

enhanced to 34.1 and 5.6 h but the t1/2 of S81Q was dramatically reduced at 70 and 80°C,

307

respectively. The structural stability of S81A was further investigated by Nano-DSC

308

(Figure S4). Compared with the wild-type enzyme, the Tm value of S81A was increased by

309

approximately 3°C. As illustrated in Figure 5A, the valine of the 421 position contains two

310

methyl groups that are closest to the S81 residue containing a hydroxyl located in the α-

311

helix of the C-terminal. Thus, when serine-81 was replaced with a hydrophobic residue of

312

alanine containing a methyl group, a hydrophobic interaction was formed between alanine-

313

81 and valine-421, which contributed to strengthening the locking force of the overall

314

structure and thereby enhancing the structural thermostability (Figure 5B).

315 316

Bioconversion of D-Allulose to D-Allose. The production of D-allose was investigated in 17



Page 18 of 36

317

5 mL reaction mixtures containing 25 g/L D-allulose using wild-type enzyme and mutant

318

V48N/G59N/I63N/F335S. As shown in Figure 6, the isomerization reaction of mutant and

319

wild-type enzyme approached, respectively, equilibrium at 16 and 24 h with an

320

approximately

321

V48N/G59N/I63N/F335S exhibits higher catalytic efficiency than wild-type enzyme under

322

the same reaction conditions. Moreover, D-altrose as a potential byproduct has not been

323

detected in reaction mixtures of wild-type enzyme and mutant by HPLC analysis (data not

324

shown), which can simplify the separation and purification and better for industrial

325

production of D-allose. Compared with the wild-type enzyme, the mutant

326

V48N/G59N/I63N/F335S has a better catalytic behavior in the industrial production of D-

327

allose. The D-ribose-5-phosphate isomerase from Thermotoga lettingae TMO converts D-

328

allulose to D-allose with a ratio of 32% but exhibits a low productivity. The D-galactose-

329

6-phosphate isomerase from Lactococcus lactis and glucose-6-phosphate isomerase from

330

Pyrococcus furiosus produce D-allose with 25% and 32% conversion rates but with a

331

detectable by-product, respectively.11 Compared with these D-allose-producing enzymes,

332

the C. obsidiansis OB47 L-RI displays a larger application potential.

32%

conversion

ratio.

It

is

observed

that

the

mutant

333 334

ASSOCIATED CONTENT

335

Supporting Information

336

Figure S1. SDS-PAGE analysis of mutants. Figure S2. Ramachandran plot of the C. 18


Page 19 of 36


337

obsidiansis OB47 L-RI model. Figure S3. Multiple sequence alignment of various L-RIs.

338

Figure S4. Nano DSC analysis of wild-type enzyme (A) and V48N/G59N/I63N/F335S

339

mutant (B). Table S1. Primers for site-directed mutagenesis.

340 341

AUTHOR INFORMATION

342

Corresponding Authors

343

* (W. Mu) Phone: +86 510 85919161. Fax: +86 510 85919161. E-mail:

344

[email protected].

345

Funding

346

This work was supported by the Support Project of Jiangsu Province (No. 2015-SWYY-

347

009), the Research Program of State Key Laboratory of Food Science and Technology,

348

Jiangnan University (No. SKLF-ZZA-201802 and SKLF-ZZB-201814), and the National

349

First-Class Discipline Program of Food Science and Technology (No. JUFSTR20180203).

350

Ethical Statement

351

This article does not contain any studies with human participants performed by any of the

352

authors.

353

19



354 355 356 357 358

REFERENCES (1) Izumori, K., Bioproduction strategies for rare hexose sugars. Naturwissenschaften. 2002, 89 (3), 120-124. (2) Iga, Y.; Nakamichi, K.; Shirai, Y., Acute and sub-chronic toxicity of D-allose in rats. Biosci., Biotechnol., Biochem. 2010, 74 (7), 1476-1478.

359

(3) Mooradian, A. D.; Smith, M.; Tokuda, M., The role of artificial and natural sweeteners

360

in reducing the consumption of table sugar: A narrative review. Clin. Nutr. Espen. 2017,

361

1-8.

362

(4) Noguchi, C.; Kamitori, K.; Hossain, A.; Hoshikawa, H.; Katagi, A.; Dong, Y.; Sui, L.;

363

Tokuda, M.; Yamaguchi, F., D-Allose inhibits cancer cell growth by reducing GLUT1

364

expression. Tohoku J. Exp. Med. 2016, 238 (2), 131.

365 366

(5) Sui, L.; Nomura, R.; Dong, Y.; Yamaguchi, F.; Izumori, K.; Tokuda, M., Cryoprotective effects of D-allose on mammalian cells. Cryobiology. 2007, 55 (2), 87.

367

(6) Gao, D.; Kawai, N.; Nakamura, T.; Lu, F.; Fei, Z.; Tamiya, T., Anti-inflammatory

368

effect of D-allose in cerebral ischemia/reperfusion injury in rats. Neurol. Med-Chir. 2013,

369

53 (6), 365-374.

370

(7) Yamada, K.; Noguchi, C.; Kamitori, K.; Dong, Y.; Hirata, Y.; Hossain, M. A.;

371

Tsukamoto, I.; Tokuda, M.; Yamaguchi, F., Rare sugar D-allose strongly induces

372

thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells. Nutr.

373

Res. 2012, 32 (2), 116-123. 20


Page 20 of 36

Page 21 of 36


374

(8) Shinohara, N.; Nakamura, T.; Abe, Y.; Hifumi, T.; Kawakita, K.; Shinomiya, A.;

375

Tamiya, T.; Tokuda, M.; Keep, R. F.; Yamamoto, T.; Kuroda, Y., D-Allose attenuates

376

overexpression of inflammatory cytokines after cerebral ischemia/reperfusion injury in

377

gerbil. J. Stroke. Cerebrovasc. 2016, 25 (9), 2184-2188.

378

(9) Kimura, S.; Zhang, G. X.; Nishiyama, A.; Nagai, Y.; Nakagawa, T.; Miyanaka, H.;

379

Fujisawa, Y.; Miyatake, A.; Nagai, T.; Tokuda, M.; Abe, Y., D-Allose, an all-cis aldo-

380

hexose, suppresses development of salt-induced hypertension in Dahl rats. J. Hypertens.

381

2005, 23 (10), 1887-1894.

382

(10) Hossain, M. A.; Wakabayashi, H.; Goda, F.; Kobayashi, S.; Maeba, T.; Maeta, H.,

383

Effect of the immunosuppressants FK506 and D-allose on allogenic orthotopic liver

384

transplantation in rats. Transplant. Proc. 2000, 32 (7), 2021-2023.

385

(11) Chen, Z.; Chen, J.; Zhang, W.; Zhang, T.; Guang, C.; Mu, W., Recent research on

386

the physiological functions, applications, and biotechnological production of D-allose.

387

Appl. Microbiol. Biotechnol. 2018, 1-10.

388 389

(12) Herber, R. R.; Maher, G. F.; Arnold, E. C.; Lorsbach, T. W., Preparation of high purity D-allose from D-glucose. US Patent No. 5433793.

390

(13) Leang, K.; Takada, G.; Fukai, Y.; Morimoto, K.; Granström, T. B.; Izumori, K.,

391

Novel reactions of L-rhamnose isomerase from Pseudomonas stutzeri and its relation with

392

D-xylose isomerase via substrate specificity. Biochim. Biophys. Acta, Gen. Subj. 2004,

393

1674 (1), 68-77. 21



394

(14) Seo, M. J.; Choi, J. H.; Kang, S. H.; Shin, K. C.; Oh, D. K., Characterization of L-

395

rhamnose isomerase from Clostridium stercorarium and its application to the production

396

of D-allose from D-allulose (D-psicose). Biotechnol. Lett. 2017, 1-10.

397

(15) Xu, W.; Zhang, W.; Tian, Y.; Zhang, T.; Jiang, B.; Mu, W., Characterization of a

398

novel thermostable L-rhamnose isomerase from Thermobacillus composti KWC4 and its

399

application for production of D-allose. Process Biochem. 2017, 53, 153-161.

400

(16) Bai, W.; Shen, J.; Zhu, Y.; Men, Y.; Sun, Y.; Ma, Y., Characteristics and kinetic

401

properties of L-rhamnose isomerase from Bacillus subtilis by isothermal titration

402

calorimetry for the production of D-allose. Food Sci. Technol. Res. 2015, 21 (1), 13-22.

403

(17) Kim, Y. S.; Shin, K. C.; Lim, Y. R.; Oh, D. K., Characterization of a recombinant L-

404

rhamnose isomerase from Dictyoglomus turgidum and its application for L-rhamnulose

405

production. Biotechnol. Lett. 2013, 35 (2), 259-264.

406

(18) Poonperm, W.; Takata, G.; Okada, H.; Morimoto, K.; Granstrom, T. B.; Izumori, K.,

407

Cloning, sequencing, overexpression and characterization of L-rhamnose isomerase from

408

Bacillus pallidus Y25 for rare sugar production. Appl. Microbiol. Biotechnol. 2007, 76 (6),

409

1297-1307.

410

(19) Park, C. S.; Yeom, S. J.; Lim, Y. R.; Kim, Y. S.; Oh, D. K., Characterization of a

411

recombinant thermostable L-rhamnose isomerase from Thermotoga maritima ATCC

412

43589 and its application in the production of L-lyxose and L-mannose. Biotechnol. Lett.

413

2010, 32 (12), 1947-1953. 22


Page 22 of 36

Page 23 of 36


414

(20) Lin, C. J.; Tseng, W. C.; Fang, T. Y., Characterization of a thermophilic L-rhamnose

415

isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494. J. Agric. Food Chem.

416

2011, 59 (16), 8702-8708.

417

(21) Takata, G.; Uechi, K.; Taniguchi, E.; Kanbara, Y.; Yoshihara, A.; Morimoto, K.;

418

Izumori, K., Characterization of Mesorhizobium loti L-rhamnose isomerase and its

419

application to L-talose production. Biosci., Biotechnol., Biochem. 2011, 75 (5), 1006-1009.

420

(22) Lin, C. J.; Tseng, W. C.; Lin, T. H.; Liu, S. M.; Tzou, W. S.; Fang, T. Y.,

421

Characterization of a thermophilic L-rhamnose isomerase from Thermoanaerobacterium

422

saccharolyticum NTOU1. J. Agric. Food Chem. 2010, 58 (19), 10431-10436.

423

(23) Korndörfer, I. P.; Fessner, W. D.; Matthews, B. W., The structure of rhamnose

424

isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change

425

between inter and intra-subunit complementation during evolution. J. Mol. Biol. 2000, 300

426

(4), 917-933.

427

(24) Doan, T. N.; Prabhu, P.; Kim, J. K.; Ahn, Y. J.; Natarajan, S.; Kang, L. W.; Park, G.

428

T.; Lim, S. B.; Lee, J. K., Crystallization and preliminary X-ray crystallographic analysis

429

of L-rhamnose isomerase with a novel high thermostability from Bacillus halodurans.

430

Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2010, 66, 677-680.

431

(25) Yoshida, H.; Yamada, M.; Ohyama, Y.; Takada, G.; Izumori, K.; Kamitori, S., The

432

structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-

433

rhamnose and D-allose provide insights into broad substrate specificity. J. Mol. Biol. 2007, 23



434

365 (5), 1505-1516.

435

(26) Yoshida, H.; Takeda, K.; Izumori, K.; Kamitori, S., Elucidation of the role of Ser329

436

and the C-terminal region in the catalytic activity of Pseudomonas stutzeri L-rhamnose

437

isomerase. Protein Eng., Des. Sel. 2010, 23 (12), 919-927.

438

(27) Chen, Z.; Xu, W.; Zhang, W.; Zhang, T.; Jiang, B.; Mu, W., Characterization of a

439

thermostable recombinant L-rhamnose isomerase from Caldicellulosiruptor obsidiansis

440

OB47 and its application for the production of L-fructose and L-rhamnulose. J. Sci. Food

441

Agric. 2017, 98 (6), 2184-2193.

442

(28) Xu, W.; Zhang, W. L.; Zhang, T.; Jiang, B.; Mu, W. M., L-Rhamnose isomerase and

443

its use for biotechnological production of rare sugars. Appl. Microbiol. Biotechnol. 2016,

444

100 (7), 2985-2992.

445

(29) Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T., The SWISS-MODEL workspace: a

446

web-based environment for protein structure homology modelling. Bioinformatics 2006,

447

22 (2), 195-201.

448 449

(30) Kiefer, F.; Arnold, K.; Künzli, M.; Bordoli, L.; Schwede, T., The SWISS-MODEL Repository and associated resources. Nucleic Acids Res. 2008, 37, 387-392.

450

(31) Biasini, M.; Bienert, S.; Waterhouse, A.; Arnold, K.; Studer, G.; Schmidt, T.; Kiefer,

451

F.; Cassarino, T. G.; Bertoni, M.; Bordoli, L., SWISS-MODEL: modelling protein tertiary

452

and quaternary structure using evolutionary information. Nucleic Acids Res 2014, 42, 252-

453

258. 24


Page 24 of 36

Page 25 of 36

454 455 456 457


(32) Bowie, J. U.; Luthy, R.; Eisenberg, D., A method to identify protein sequences that fold into a known three-dimensional structure. Science. 1991, 253 (5016), 164-170. (33) Lüthy, R.; Bowie, J. U.; Eisenberg, D., Assessment of protein models with threedimensional profiles. Nature. 1992, 356 (6364), 83.

458

(34) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M., PROCHECK: a

459

program to check the stereochemical quality of protein structures. J. Appl. Crystallogr.

460

1993, 26 (2), 283-291.

461

(35) Xu, W.; Ni, D.; Yu, S.; Zhang, T.; Mu, W., Insights into hydrolysis versus

462

transfructosylation: Mutagenesis studies of a novel levansucrase from Brenneria sp.

463

EniD312. Int. J. Biol. Macromol. 2018, 116, 335-345.

464 465 466 467 468 469

(36) Laederach, A.; Reilly, P. J., Specific empirical free energy function for automated docking of carbohydrates to proteins. Int. J. Biol. Macromol. 2003, 24 (14), 1748-1757. (37) Rosebrough, N. J.; Farr, S. A.; Randall, R. L.; Lowry, O. H., Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193 (1), 265. (38) Dische, Z.; Borenfreund, E., A new spectrophotometric method for the detection and determination of keto sugars and trioses. J. Biol. Chem. 1951, 192 (2), 583-587.

470

(39) Bendl, J.; Stourac, J.; Sebestova, E.; Vavra, O.; Musil, M.; Brezovsky, J.; Damborsky,

471

J., HotSpot Wizard 2.0: automated design of site-specific mutations and smart libraries in

472

protein engineering. Nucleic Acids Res. 2016, 44, 479-487.

25



473

Figure legend

474

Figure 1. C. obsidiansis OB47 L-RI structural model and alignment with B. halodurans

475

ATCC BAA-125 L-RI (PDB ID: 3P14). (A) Dimer model of C. obsidiansis OB47 L-RI.

476

The α-helix, β-strand and random coil were respectively colored cyan, magenta and salmon.

477

(B) Monomer superimposition of C. obsidiansis OB47 L-RI (cyan) and B. halodurans

478

ATCC BAA-125 L-RI (warm pink).

479 480

Figure 2. Planform (A) and lateral view (B) of the surface model of wild-type enzyme.

481

Surface and cartoon models were respectively delineated cyan and green. The β1-α1-loop

482

(raspberry) embraced the catalytic pocket (yellow line) and partly covered the catalytic

483

tunnel (green). Superimposition of surface and cartoon models by part (C) and whole (D)

484

transparency.

485 486

Figure 3. Residue distributions of 48, 59, 63 and 335 positions of wild-type enzyme (A)

487

and V48N/G59N/I63N/F335S mutant (B). These residues were presented as stick models.

488 489

Figure 4. Surface models of wild-type enzyme (A) and V48N/G59N/I63N/F335S mutant

490

(B). Residues of 48, 59, 63 and 335 positions were respectively colored magenta, orange,

491

blue and green.

492 26


Page 26 of 36

Page 27 of 36


493

Figure 5. Location of S81 and V421 in a cartoon model of wild-type enzyme (A) and

494

V48N/G59N/I63N/F335S mutant (B). The hydrophobic interaction was represented using

495

red dotted lines.

496 497

Figure 6. Production of D-allose using the V48N/G59N/I63N/F335S mutant and wild-type

498

enzyme. The conversion reactions were carried out at 60°C and pH 8.0 containing 1 mM

499

Co2+, 5 μM of purified enzyme and 25 g/L D-allulose as substrate. The experiments were

500

conducted in three replications ± standard deviation.

27



Page 28 of 36

Table 1. Relative activities towards L-rhamnose and D-allulose of wild-type enzyme and mutants Enzymes

Relative activity (%) L-rhamnose

D-allulose

Wild-type

100.0 ± 2.2

100.0 ± 1.8

V48N

49.9 ± 0.8

168.6 ± 1.6

G59N

86.0 ± 0.4

161.4 ± 1.2

G60T

86.5 ± 0.6

87.1 ± 0.6

G62T

70.4 ± 0.6

136.1 ± 1.4

I63N

40.8 ± 0.8

94.5 ± 1.1

I101N

87.4 ± 1.1

136.8 ± 1.2

F335C

69.6 ± 1.6

187.4 ± 2.0

F335S

59.1 ± 1.4

131.7 ± 2.1

V48N/ G59N/ I63N

38.5 ± 0.7

205.6 ± 2.4

V48N/ G59N/ I63N/F335S

39.4 ± 0.5

234.1 ± 2.2

28


Page 29 of 36


Table 2. Thermostability of C.obsidiansis OB47 L-RI mutations Enzymes

Half-life t1/2（h） 70°C

80°C

Wild-type

26.4

4.5

S81A

34.1

5.6

S81Q

6.5

1.8

S88R

5.7

2.0

I343A

4.7

1.9

V421I

13.6

4.6

29



Figure 1. C. obsidiansis OB47 L-RI structural model and alignment with B. halodurans ATCC BAA-125 L-RI (PDB ID: 3P14). (A) Dimer model of C. obsidiansis OB47 L-RI. The α-helix, β-sheet and random coil were respectively colored cyan, magenta and salmon. (B) Monomer superimposition of C. obsidiansis OB47 L-RI (cyan) and B. halodurans ATCC BAA-125 L-RI (warm pink). 99x49mm (300 x 300 DPI)


Page 30 of 36

Page 31 of 36


Figure 2. Planform (A) and lateral view (B) of the surface model of wild-type enzyme. Surface and cartoon models were respectively delineated cyan and green. The β1-α1-loop (raspberry) embraced the catalytic pocket (yellow line) and partly covered the catalytic tunnel (green). Superimposition of surface and cartoon models by part (C) and whole (D) transparency. 88x86mm (300 x 300 DPI)



Figure 3. Residue distributions of 48, 59, 63 and 335 positions of wild-type enzyme (A) and V48N/G59N/I63N/F335S mutant (B). These residues were presented as stick models. 91x50mm (300 x 300 DPI)


Page 32 of 36

Page 33 of 36


Figure 4. Surface models of wild-type enzyme (A) and V48N/G59N/I63N/F335S mutant (B). Residues of 48, 59, 63 and 335 positions were respectively colored magenta, orange, blue and green. 99x51mm (300 x 300 DPI)



Figure 5. Location of S81 and V421 in a cartoon model of wild-type enzyme (A) and V48N/G59N/I63N/F335S mutant (B). The hydrophobic interaction was represented using red dotted lines. 82x40mm (300 x 300 DPI)


Page 34 of 36

Page 35 of 36


Figure 6. Production of D-allose using the V48N/G59N/I63N/F335S mutant and wild-type enzyme. The conversion reactions were carried out at 60°C and pH 8.0 containing 1 mM Co2+, 5 μM of purified enzyme and 25 g/L D-allulose as substrate. The experiments were conducted in three replications ± standard deviation. 271x189mm (300 x 300 DPI)



TOC graphic 84x47mm (300 x 300 DPI)


Page 36 of 36

Improving Thermostability and Catalytic Behavior of l-Rhamnose

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