A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

doi:10.21203/rs.3.rs-24804/v2

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A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

https://doi.org/10.21203/rs.3.rs-24804/v2

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published 04 Aug, 2020

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Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H}, and one negative variant TrCel1b^N240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1b^{I177S/I174S/W173H}was accelerated and then the maximum total production (195.8 mg/mL/mg enzyme) of the synthesized disaccharides was increased by 3.5-fold compared to that of wildtype. On the contrary, TrCel1b^N240I lost reverse hydrolysis activity. The results demonstrate that the average hydropathy index of the key amino acid residues in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

General Biochemistry

β-glucosidase

hydropathy index

disaccharide synthesis

reverse hydrolysis reaction

site-directed mutagenesis

Oligosaccharides are widely distributed in nature and are used in the food and medical industries (Wang et al. 2019; Wen et al. 2018; Driguez et al. 2014; Lu et al. 2015; McCranie et al. 2014; Schmaltz et al. 2011). Oligosaccharides are mainly prepared by extraction and isolation from a variety of natural plants, by chemical synthesis or biosynthesis (Wen et al. 2018; Perugino et al. 2004; Cheng et al. 2019). Extraction from plants is limited by the source plant and its terrestrial distribution. The biosynthesis of oligosaccharides via enzymatic synthesis technique in vitro has recently received increasing attention due to attributes including mild reaction temperature and excellent regio- and stereo-selectivity without the need for masking of functional groups (Moracci et al. 2001). Enzymatic synthesis of oligosaccharides is mainly catalyzed by glycosidases or glycosyltransferases (Perugino et al. 2004). Synthesis of oligosaccharides by glycosidases has many advantages that include of simplicity, reliability, ease of operation, and broad substrate specificity (Cobucci‐Ponzano et al. 2011; Salgado et al. 2018), compared with catalysis by glycosyltransferases, which is the absolute conservation on the substrate (Moremen et al. 2019). These facts favor glycosidase as an economically feasible approach in the production of oligosaccharides (García et al. 2017). Furthermore, some high value-added oligosaccharides, such as laminaribiose, gentiobiose, and sophorose, have been produced using glycosidases (da Silva et al. 2017; Semenova et al. 2015). These oligosaccharides reportedly have potential applications in food and enzyme industries (Sanz et al. 2005; Xia et al. 2018).

In addition to glycosidic bond cleavage, glycoside hydrolases (GHs) can be used for the synthesis of glycoside bonds in vitro via reverse hydrolysis reaction without the need for cofactors, such as uridine diphosphate (Lu et al. 2015; Perugino et al. 2004; Rosengren et al. 2019; Xu et al. 2016). Recently, several glycosidases, such as β-glucosidase, α-glucosidase, α-_L-rhamnosidase, β-mannosidase and β-galactosidase, have been used to synthesize glycosides via the reverse hydrolysis reaction (Lu et al. 2015; Arthornthurasuk et al. 2018; Chen et al. 2019; Ajisaka et al. 2012; Tan et al. 2016). Gentiooligosaccharides, octyl glucoside, laminaribiose, sophorose, cellobiose, and gentiobiose have been synthesized by β-glucosidase via reverse hydrolysis reaction (Semenova et al. 2015; Arthornthurasuk et al. 2018; Wang et al. 2018; Ravet et al. 1993). Furthermore, protein engineering of GHs has been widely used to eliminate hydrolytic activity and improve synthetic activity. An important strategy is to disrupt the binding of catalytic water. Honda et al. (2008) reported that hydrogen-bonding interaction with catalytic water that reduced the hydrolytic reactivity of an inverting xylanase was dramatically decreased by eliminating the retention of the nucleophilic water molecule at the key amino acid residue. Other studies have focused on improving the hydrophobicity of the entrance to the active site (Xu et al. 2016; Qin et al. 2019; Frutuoso et al. 2013) or acceptor subsite (Lundemo et al. 2013). However, the effect of hydrophobicity of amino acid residues in the catalytic site on the catalytic activity needs to be further elucidated.

In this study, we report the ability of β-glucosidase TrCel1b from Trichoderma reesei to simultaneously catalyze the synthesis of three disaccharides (laminaribiose, sophorose, and cellobiose) from glucose. The three-dimensional structure of TrCel1b was obtained and docked with cellobiose as the model of disaccharides. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity.

To verify our deduction, the Hydropathy Index For Enzyme Activity (HIFEA) strategy was devised. Three hydrophobic amino acid residues in the catalytic site were mutated into hydrophilic residues, which generated the maximal change in the hydropathy index. Thus, three variants were obtained: TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H}. Additionally, the variant TrCel1b^N240I was obtained by improving the hydrophobicity in the catalytic site. The production of synthesized disaccharides by the three variants were investigated. Total production (195.8 mg/mL/mg enzyme) of the synthesized disaccharides was increased by 3.5 times, compared to that of the wild type. Especially, the production of laminaribiose and sophorose reached 92.3 and 71.1 mg/mL/mg enzyme. The findings indicate the value of the HIFEA strategy in providing a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

Chemicals, plasmids, and culture media

Laminaribiose, sophorose, p-nitrophenol (pNP), and p-nitrophenol-β-_D-glucoside(pNPG) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Kanamycin and isopropyl-1-thio-β-_D-galactopyranoside (IPTG) were purchased from Gen-view Scientific Inc. (El Monte, CA, USA). The KOD-Plus-Mutagenesis Kit was purchased from Toyobo Co., Ltd. (Osaka, Japan). All other chemicals were from Sangon Biotech Co., Ltd. (Shanghai, China). Plasmid pET-32a was purchased from Invitrogen (Carlsbad, CA, USA). Restriction enzymes and T4 DNA ligase were purchased from Thermo Fisher Scientific (Shanghai, China). Primers were synthesized by Sangon Biotech Co., Ltd. Escherichia coli DH5α and E. coli BL21 (DE3) was purchased from TransGen Biotech (Beijing, China).

Site-directed mutagenesis

TrCel1b (GenBank accession number: AAP57758.1) was amplified from the cDNA of T. reesei QM6a (ATCC 13631) with primers harboring EcoRΙ and HindⅢ sites, and ligated into pET-32a after it was digested with the same enzymes to obtain the recombinant vector pET-32a-WT. The recombinant vectors pET-32a-I177S, pET-32a-I177S/I174S, pET-32a-I177S/I174S/W173H, and pET-32a-N240I were constructed using the aforementioned KOD-Plus-Mutagenesis Kit. Oligonucleotides used in this study for plasmid constructions are listed in Supplementary Table S2.

Expression and purification

The constructed vectors were introduced into E. coli BL21(DE3) for protein expression and transformants were selected on LB plates containing 10 μg/mL ampicillin as previously described (Xin et al. 2016). These proteins were purified from the supernatant with His SpinTrap columns (GE Healthcare, Uppsala, Sweden) as previously described (Hou et al. 2019). The purified protein of TrCel1b and its variants was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described (Schagger 2006).

Measurement of β-glucosidase activity and enzymatic synthesis of disaccharides

β-glucosidase activity was measured using 50 µL of 5 mM p-nitrophenyl-β-_D-glucopyranoside (pNPG) as substrate at 30 ^oC for 30 min as previously described method (Liu et al. 2016). The unit of β-glucosidase activity was defined as the amount of enzyme required to release total reducing sugar equivalent to 1 µmol pNP per min. The reverse hydrolysis reaction was performed in 10 ml solution containing 3.5 mg TrCel1b or its variants, 1-8 g glucose, 500 μL glycerol, and 10 mg sodium azide in 50 mM phosphate buffer or 0.2 M sodium phosphate dibasic and 0.1 M citric acid buffer and the reaction solution was kept under 30 °C. The solution was sampled at specific time and three independent replicates were carried out. The sample was diluted 1/4 - 1/2 with purified water before the preparation sample was analyzed by HPLC that was equipped with a refractive index detector (Hitachi, Tokyo, Japan) using an Inertsil NH₂ column at 45 °C (250 mm × 7.8 mm; Shimadzu, Kyoto, Japan). The mobile phase was 80% acetonitrile and a flow rate was 1.0 mL/min. The fractions containing disaccharides were collected and the laminaribiose, sophorose, and cellobiose were further identified with Thin layer chromatography (TLC) using aluminum-backed sheets of silica gel 60F₂₅₄ (0.2 mm thick; E. Merck, Germany). Elution was carried out with n-butanol: ethanol: water (5:3:2). The plates were visualized by exposure to staining solution containing 3 g phenol, 5 mL concentrated sulfuric acid and 95 mL alcohol followed by charring. Thermodynamic parameters for the reverse hydrolysis reaction were calculated from glucose to laminaribiose, sophorose, and cellobiose at pH 7.4 with an ionic strength of 0.05 M using the eQuilibrator (Noor et al. 2013).

The phylogenetic and structural analysis

The phylogenetic tree of TrCel1b (GenBank accession no. EGR49111.1) from T. reesei QM6a, ThBgl2 (5JBO) from T. harzianum, ThBgl1 (5JBK) from T. harzianum, HiBG (AII80277.1) from Humicola insolens, NkBgl (BAB91145.1) from Neotermes koshunensis and CaBglA (JX030398.1) from Caldicellulosiruptor sp. F32 was generated using MEGA. The three-dimensional structures of TrCel1b, TrCel1b^I177S, TrCel1b^I177S/I174S, TrCel1b^{I177S/I174S/W173H}, and TrCel1b^N240Iwere predicted using SWISS-MODEL (Waterhouse et al. 2018) and I-TASSEA (Yang et al. 2015) with the crystal structure of TrCel1b-H13 (PDB ID: 6KHT) as the template. The structures were illustrated using PyMOL software (Delano Scientific, Palo Alto, CA). The interaction between protein and cellobiose was analyzed by LigPlot and Discovery Studio Software (Accelrys, San Diego, CA, USA).

Statistics

The Student’s t-test was performed for significant differences between two groups of data. P<0.05 was considered statistically significant and standard deviations (SD) were calculated at least in triplicate.

The rational design of TrCel1b

TrCel1b (GenBank no. EGR49111.1) in T. reesei belongs to the GH1 family and shares 92, 52, 52, 39, and 38% amino acid sequence identity with GH1 family β-glucosidase ThBgl2 (5JBO), ThBgl1 (5JBK), HiBG (AII80277.1), NkBgl (BAB91145.1), and CaBglA (JX030398.1), respectively. Mature TrCel1b consists of 484 amino acids with a molecular weight of 55.1 kDa. As shown in Fig. S1, β-glucosidases ThBgl1 and ThBgl2, which have a relatively close evolutionary relationship with TrCel1b, synthesize oligosaccharides (Florindo et al. 2018). The three-dimensional structure of TrCel1b was predicted based on the structure of its variant (PDB ID: 6KHT) by SWISS-MODEL and I-TASSER (Fig. S2). The sequence of TrCel1b shared 100% identity with that of 6KHT (Coverage: 98%). A typical TIM-barrel structure of (α/β)₈ fold was evident in TrCel1b (Fig. 2A), as well as the other GH1 family members, such as β-glucosidase ThBgl2.

The reverse hydrolysis reaction was performed with purified TrCel1b in 50 mM phosphate buffer at pH 7.4 under 30 ^oC. The results of TLC and HPLC indicate that the three disaccharides - laminaribiose, sophorose, and cellobiose were simultaneously produced through the reverse hydrolysis reaction with inexpensive glucose as the glucosyl donor (Fig. 1 and Fig. S3). Moreover, the production of laminaribiose and sophorose was similar, with the production of cellobiose being markedly lower (Fig. S4 A-L, A-S, A-C).

To improve the production of disaccharides, interaction between TrCel1b and cellobiose as the model of disaccharide was predicted with Autodock 1.5.6 (Fig. 2B). The amino acid residues surrounded by the glucose moiety in aglycone subsite (+1 subsite) reportedly have a significant effect on synthesis capacity compared to the residues surrounded by the glucose moiety of cellobiose at the -1 subsite (Frutuoso et al. 2013; Lundemo et al. 2013; Florindo et al. 2018; Lundemo et al. 2017). The glucose moiety of cellobiose in the aglycone subsite is surrounded by two hydrophobic residues (W173 and I174) and two hydrophilic residues (Y178 and N240), within a distance of 3.1 Å. The predicted distances between W173, I174, Y178, or N240 and cellobiose were 3.1, 2.8, 2.1, and 2.1 Å, respectively. The extremely hydrophobic residue I177 was also found at the +2 subsite at a distance of 6.4 Å. The hydropathy index of I, W, Y, and N was 4.5, −0.9, −1.3, and −3.5, respectively.

To verify our hypothesis, the HIFEA strategy was applied to improve the reverse hydrolysis activity by reducing the average hydropathy index of key amino acid residues in catalytic site (I_ah) of TrCel1b. I_ah was defined as the sum of the hydropathy index of amino acid residues 173, 174, 177, and 240 divided by their number, namely, I_ah = (I _{h, 173} + I _{h, 174}+ I _{h, 177} + I_{h, 240}) / 4. Three hydrophobic amino acid residues in the catalytic site were mutated into hydrophilic residues and three variants TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H} were obtained. Additionally, the variant TrCel1b^N240I was obtained by improving the hydrophobicity in the catalytic site. The I_ah of TrCel1b and its variants was calculated according to the hydropathy index. The I_ahof TrCel1b, TrCel1b^I177S, TrCel1b^I177S/I174S, TrCel1b^{I177S/I174S/W173H}, and TrCel1b^N240I was 1.15, −0.175, −1.5, −2.075, and 3.15, respectively, which revealed that I_ahchanged along with the mutation. The hydrophobic interaction between these key amino acid residues and disaccharides was weakened when I_ah was reduced by the mutation of the hydrophobic residues located in the catalytic site of W173, I174, and/or I177 to the hydrophilic residues, which facilitated the release of disaccharide. Finally, production of disaccharides synthesized by reverse hydrolysis was improved. On the contrary, reverse hydrolysis was repressed when I_ah was increased by improving the hydrophobicity in the catalytic site.

Identification of products synthesized by TrCel1b wildtype and its variants

Purified TrCel1b^I177S, TrCel1b^I177S/I174S, TrCel1b^{I177S/I174S/W173H}, and TrCel1b^N240I displayed a similar molecular weight of 74 kDa, compared to the size of TrCel1b (Fig. S5), consistent with the molecular weight predicted by the ExPASy website (https://web.expasy.org/compute_pi/). The products synthesized by these variants, except TrCel1b^N240I, were laminaribiose, sophorose, and cellobiose, respectively (Fig. 1, lane 7–9). The hydrolysis activities of the variants decreased as the I_ah of variants decreased (Fig. 3A) and β-glucosidase activity of TrCel1b^{I177S/I174S/W173H} was almost lost, compared to 0.35 U/mg soluble protein of TrCel1b. However, there was no significant change of β-glucosidase activity between TrCel1b and TrCel1b^N240I. The production of disaccharides was enhanced as the I_ah of the variants decreased (Fig. 3B). The disaccharide production of TrCel1b^{I177S/I174S/W173H} increased by 3.5 times, reaching 68.5 mg/mL, compared with that of TrCel1b. On the contrary, the variant TrCel1b^N240I with an I_ah of 3.15 displayed no disaccharide synthetic activity. As shown in Fig. S4, the disaccharides laminaribiose, sophorose, and cellobiose were synthesized by TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H} the same as that of TrCel1b. Moreover, the production of laminaribiose, sophorose, and cellobiose synthesized by TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H} were greater than those of TrCel1b (Fig. S4). These results indicated that the decreased I_ah of TrCel1b was favorite for the reverse hydrolysis reaction.

Glucose was used as the substrate (10, 20, 40, 60, and 80%) for reverse hydrolysis and the laminaribiose, sophorose and cellobiose production was measured by high-performance liquid chromatography (HPLC) (Fig. S4). The production of laminaribiose, sophorose, and cellobiose was greatly improved as glucose concentration increased from 10% to 80%. Moreover, we investigated the effects of pH value on synthesis activity in 0.2 M sodium phosphate dibasic and 0.1 M citric acid buffer (Fig. S6), and then it was proved that the optimal pH for TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H}were 7, 6, and 6, respectively. The activity of TrCel1b^I177S/I174S and TrCel1b^{I177S/I174S/W173H} increases by 55% and 46% in comparison with TrCel1b at pH 6 (Fig. S6). When the hydrophobic residue isoleucine at the +1 subsite of TrCel1b is mutated to hydrophilic residue serine, the reverse hydrolysis activity of TrCel1b is improved; when W173 at +1 subsite of TrCel1b is mutated to histidine, protonation of histidine may occur under the weakly acidic environment, which is unfavorable to reverse hydrolysis activity (Lundemo et al. 2017; Seidle et al. 2005). Thus, the total disaccharides production synthesized by TrCel1b^{I177S/I174S/W173H} is slightly lower than that of TrCel1b^I177S/I174S at pH 6 (Fig. S6).

The production of laminaribiose and sophorose was increased when the I_ah value of the variants was decreased. Laminaribiose production was the highest among the three disaccharides. The maximal laminaribiose production by TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H} reached 20.0, 27.6, 32.3 mg/mL, and increased 1.8, 2.8, and 3.5-fold, compared to that of TrCel1b, respectively (Fig. S4). Sophorose production was markedly higher than that of cellobiose, and its maximal production of TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H} reached 17.8, 19.9, and 24.9 mg/mL, respectively (Fig. S4). These results revealed a direct relationship between the reverse hydrolysis activity and the I_ah value of TrCel1b.

As shown in Fig. 3B, the increased disaccharides production from TrCel1b^I177S to TrCel1b^I177S/I174 is more than two times higher than that from TrCel1b^I177S/I174S to TrCel1b^{I177S/I174/W173H}. As shown in Fig. 2B, I177 is located in +2 site and then W173 and I174 are located in +1 site. Our results were consistent with previous report that the position of amino acid residue as well as I_ah value has an important influence on the disaccharides synthesis (Rosengren et al., 2014). We speculated that I177 has a more significant influence on the entrance of glucose and the release of disaccharides, compared to W173 and I174 owe to +2 site is closer to the entrance of enzyme, compared to +1 site.

To study the effect of mutation on the interaction between protein and disaccharide, cellobiose was docked with TrCel1b and its variants using Autodock software and the result of docking was analyzed by LigPlot. The hydrophobic interaction between cellobiose and the amino acid residues of TrCel1b^{I177S/I174S/W173H} (Fig. 4B) became weak, compared to that of TrCel1b (Fig. 4A). The number of residues that hydrophobically interacted with cellobiose in TrCel1b^{I177S/I174S/W173H} and TrCel1b was 16 and 20, respectively. The hydrophobic interactions between the W173 and I174 residues and cellobiose disappeared since the two residues were mutated into the hydrophilic residues (Fig. 4A and 4B). These results were verified using Discovery studio (Fig. S7).

The relationship between the I_ah value of TrCel1b and reverse hydrolysis activity

Compared with TrCel1b, the I_ah of TrCel1b^{I177S/I174S/W173H} decreased, which was beneficial for the release of the cellobiose product. On the contrary, the I_ah of TrCel1b^N240I was enhanced and the reverse hydrolysis activity of TrCel1b^N240I was completely lost (Fig. 3B) owing to the mutation of the hydrophilic residue N240 to the hydrophobic residue isoleucine (Fig. 4C). The findings provided an obvious indication of a direct relationship between the I_ah value of TrCel1b and reverse hydrolysis activity. When the I_ah value of TrCel1b became negative, the reverse hydrolysis activity was enhanced. On the contrary, when the I_ahvalue of TrCel1b increased, the reverse hydrolysis activity was abolished. The findings are consistent with our hypothesis that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis.

Table 1 Thermodynamic parameters for the reverse hydrolysis from glucose to laminaribiose, cellobiose, and sophorose

Process	K_eq(×10^-3)	∆_rG’⁰ (kJ/mol)	∆_rG’ (kJ/mol)
2 Glucose = Laminaribiose + H₂O	3.5	14.0 ± 6.0	-44.7 ± 6.0
2 Glucose = Cellobiose + H₂O	5.4	12.9 ± 3.7	-45.8 ± 3.7
2 Glucose = Sophorose + H₂O	3.4	14.1 ± 5.0	-44.6 ± 5.0

∆_rG’⁰: the change in Gibbs free energy of the chemical reaction in standard 1 M concentrations of substrates and products at pH 7.4 with ionic strength of 0.05 M.

K_eq: the equilibrium constant of the chemical reaction in standard 1 M concentrations of substrates and products at pH 7.4 with ionic strength of 0.05 M.

∆_rG’: the change in Gibbs free energy of the chemical reaction in 4.4 M concentrations of substrate at pH 7.4 with ionic strength of 0.05 M.

Comparison of disaccharides production synthesized by β-glucosidases

In this study, an β-glucosidase TrCel1b from T. ressei was shown to simultaneously synthesize laminaribiose, sophorose, and cellobiose using a high concentration glucose as substrate. As shown in Table 1, the ∆_rG’ of laminaribiose, sophorose, or cellobiose synthesis is <0 indicating it is realizable that laminaribiose, sophorose, and cellobiose were produced from glucose by TrCel1b. Ravet et al. (1993) reported that the disaccharides were produced by β-glucosidase derived from almonds. However, most of these disaccharides were gentiobiose, rather than laminaribiose and sophorose. This was the reason why the equilibrium constant (K_eq) of the reaction to synthesize laminaribiose, sophorose, and cellobiose (Table 1) was markedly lower than that of gentiobiose (53.8×10^-3). There are few reports on laminaribiose and sophorose synthesis, reflecting their low production. To improve the production of disaccharides synthesized by TrCel1b, protein engineering was performed using the HIFEA strategy. The production of laminaribiose, sophorose, and cellobiose synthesized by TrCel1b^{I177S/I174S/W173H}was increased 3.5, 2.6, and 3.9-fold, respectively, compared to that of TrCel1b (Fig. S4). Compared with reported β-glucosidases from different species (Table S1), the maximal production of laminaribiose and sophorose by TrCel1b^{I177S/I174S/W173H} reached 92.3 and 71.1 mg/mL/mg enzyme, respectively, and were higher than the results produced by β-glucosidases from Aspergillus niger, Corynascus sp., Penicillium verruculosum, T. reesei (Semenova et al. 2015), and almond (Ravet et al. 1993). To our knowledge, this is the highest production of laminaribiose and sophorose simultaneously synthesized by β-glucosidase.

Our results reveal that β-glucosidase TrCel1b from T. reesei simultaneously synthesized laminaribiose, sophorose, and cellobiose. Three variants (TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H}) with improved disaccharide production were obtained using the HIFEA strategy. The I_ah of β-glucosidase TrCel1b is an important factor for the production of laminaribiose, sophorose, and cellobiose. The decreased I_ah value of TrCel1b improved the synthetic activity and reduced the hydrolytic activity. The HIFEA strategy is implicated as a new avenue for the production of high value-added disaccharides.

HIFEA, Hydropathy Index For Enzyme Activity; I_ah, the average hydropathy index of key amino acid residues in catalytic site; TLC, thin layer chromatography; GHs, glycoside hydrolases.

Acknowledgments

We thank Ms. Zhifeng Li, Ms. Rui Wang, and Mr. Chengjia Zhang from State Key Laboratory of Microbial Technology for assistance in protein separation and purification. Thanks for Prof. Huifeng Jiang from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences for his contribution during Discovery Studio analyses. Thank for Prof. Luying Xun for revising the manuscript.

Authors’ contributions

K.N., Z.L., Y.F. and T.G. carried out protein mutagenesis and purification. K.N., Z.W., P.Z., D.G. carried out biochemical assays. K.N., Z.L. and X.F. carried out structural modeling and docking. Z.W. and X.F. conceived of the study. Z.D. and X.F. oversaw experimental and computational work. K.N., Z.D. and X.F. designed experiments and wrote the manuscript. X.F. coordinated the project.

Funding

This work was supported by National Key R&D Program of China (No. 2018YFA090010), Key Technologies R&D Program of Shandong Province (No. 2018GSF121021), the 111 Project (No. B16030), the State Key Laboratory of Microbial Technology Open Projects Fund and National Natural Science Foundation of China (No. 31870785 and 31570040).

Availability of data and materials

All data supporting this article’s conclusion are available.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

Part of the study was authorized by the patent ZL 201410438150.0.

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TableS2.jpg
Primers used in this study
Fig.S1.jpg
The phylogenetic tree of TrCel1b and other GH1 family β-glucosidases
Fig.S2.jpg
The 3-D model of TrCel1b predicted by SWISS-MODEL (A) and I-TASSER (B) using β-glucosidase 6KHT as template
Fig.S5.jpg
SDS-PAGE analysis of purification of TrCel1b and its variants
Fig.S7.jpg
The interaction profiles between TrCel1b (A), TrCel1bI177SI174SW173H (B) or TrCel1bN240I (C) and cellobiose analyzed by Discovery Studio 4.5, respectively
Fig.S8.jpg
the labeled original electropherogram
TableS1.jpg
The comparation of the production of laminaribiose and sophorose synthesized by β-glucosidase from different species
Fig.S6.jpg
Effects of pH value on disaccharides synthesis.
Fig.S3.jpg
Analysis of the disaccharides with HPLC
Fig.S4.jpg
The disaccharides synthesis of β-glucosidase and its variants under different concentrations of glucose
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A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

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