Previously, a α-d-fructofuranose-β-d-fructofuranose 1,2′:2,1′-dianhydride (DFA I)-forming inulin fructotransferase (IFTase), namely, SdIFTase, was identified. The enzyme does not show high performances. In this work, to improve catalytic behavior including activity and thermostability, the enzyme was modified using site-directed mutagenesis on the basis of structure. The mutated residues were divided into three groups. Those in group I are located at central tunnel including G236, A257, G281, T313, and A314S. The group II contains residues at the inner edge of substrate binding pocket including I80, while group III at the outer edge includes G121 and T122. The thermostability was reflected by the melting temperature (Tm) determined by Nano DSC. Finally, the Tm values of G236S/G281S/A257S/T313S/A314S in group I and G121A/T122L in group III were enhanced by 3.2 and 4.5 °C, and the relative activities were enhanced to 140.5% and 148.7%, respectively. The method in this work may be applicable to other DFA I-forming IFTases.Keywords: DFA I-forming inulin fructotransferase (IFTase); inulin; α-d-fructofuranose-β-d-fructofuranose 1,2′:2,1′-dianhydride (DFA I);

•Attention on mannitol production by fermentation is received in recent years.•Fermentation factors affected the mannitol yield are listed.•Mannitol applications in food, pharmaceutical and chemical industries are reviewed.Mannitol is a polyol or an alditol that is naturally found in many plants and can be produced by several microorganisms. Based on its beneficial physiological effects, mannitol is currently used as a functional sweetener in the food industry. In addition, mannitol has applications in pharmaceutical, chemical and medical industries because of its promising advantages. Mannitol can be produced by extraction, chemical synthesis, or fermentation. Certain mutants have been constructed for different purposes. In this review, we focused on recent advances in the applications and biotechnological production of mannitol.

d-Psicose is a highly valuable rare sugar because of its excellent physiological properties and commercial potential. d-Psicose 3-epimerase (DPEase) is the key enzyme catalyzing the isomerization of d-fructose to d-psicose. However, the poor thermostability and low catalytic efficiency are serious constraints on industrial application. To address these issues, site-directed mutagenesis of Tyr68 and Gly109 of the Clostridium bolteae DPEase was performed. Compared with the wild-type enzyme, the Y68I variant displayed the highest substrate-binding affinity and catalytic efficiency, and the G109P variant showed the highest thermostability. Furthermore, the double-site Y68I/G109P variant was generated and exhibited excellent enzyme characteristics. The Km value decreased by 17.9%; the kcat/Km increased by 1.2-fold; the t1/2 increased from 156 to 260 min; and the melting temperature (Tm) increased by 2.4 °C. Moreover, Co2+ enhanced the thermostability significantly, including the t1/2 and Tm values. All of these indicated that the Y68I/G109P variant would be appropriate for the industrial production of d-psicose.

Inulin fructotransferase (IFTase) is an important enzyme that produces di-d-fructofuranose 1,2′:2,3′ dianhydride (DAF III), which is beneficial for human health. Present investigations mainly focus on screening and characterizing IFTase, including catalytic efficiency and thermostability, which are two important factors for enzymatic industrial applications. However, few reports aimed to improve these two characteristics based on the structure of IFTase. In this work, a structural model of IFTase (DFA III-producing) from Arthrobacter sp. 161MFSha2.1 was constructed through homology modeling. Analysis of this model reveals that two residues, Ser-309 and Ser-333, may play key roles in the structural stability. Therefore, the functions of the two residues were probed by site-directed mutagenesis combined with the Nano-DSC method and assays for residual activity. In contrast to other mutations, single mutation of serine 309 (or serine 333) to threonine did not decrease the enzymatic stability, whereas double mutation (serine 309 and serine 333 to threonine) can enhance thermostability (by approximately 5 °C). The probable mechanisms for this enhancement were investigated.Keywords: di-d-fructofuranose 1,2′:2,3′ dianhydride (DFA III); homology modeling; inulin; inulin fructotransferase (IFTase); thermostability;

Rare sugars have recently drawn attention because of their potential applications and huge market demands in the food and pharmaceutical industries. All l-hexoses are considered rare sugars, as they rarely occur in nature and are thus very expensive. l-Hexoses are important components of biologically relevant compounds as well as being used as precursors for certain pharmaceutical drugs and thus play an important role in the pharmaceutical industry. Many general strategies have been established for the synthesis of l-hexoses; however, the only one used in the biotechnology industry is the Izumoring strategy. In hexose Izumoring, four entrances link the d- to l-enantiomers, ketose 3-epimerases catalyze the C-3 epimerization of l-ketohexoses, and aldose isomerases catalyze the specific bioconversion of l-ketohexoses and the corresponding l-aldohexoses. In this article, recent studies on the enzymatic production of various l-hexoses are reviewed based on the Izumoring strategy.

l-Rhamnose isomerase (L-RI, EC 5.3.1.14), catalyzing the isomerization between l-rhamnose and l-rhamnulose, plays an important role in microbial l-rhamnose metabolism and thus occurs in a wide range of microorganisms. It attracts more and more attention because of its broad substrate specificity and its great potential in enzymatic production of various rare sugars. In this article, the enzymatic properties of various reported L-RIs were compared in detail, and their applications in the production of l-rhamnulose and various rare sugars including d-allose, d-gulose, l-lyxose, l-mannose, l-talose, and l-galactose were also reviewed.

l-Arabinose isomerase (L-AI, EC 5.3.1.4) catalyzes the isomerization between l-arabinose and l-ribulose, and most of the reported ones can also catalyze d-galactose to d-tagatose, except Bacillus subtilis L-AI. In this article, the L-AI from the psychrotolerant bacterium Pseudoalteromonas haloplanktis ATCC 14393 was characterized. The enzyme showed no substrate specificity toward d-galactose, which was similar to B. subtilis L-AI but distinguished from other reported L-AIs. The araA gene encoding the P. haloplanktis L-AI was cloned and overexpressed in E. coli BL21 (DE3). The recombinant enzyme was purified by one-step nickel affinity chromatography . The enzyme displayed the maximal activity at 40 °C and pH 8.0, and showed more than 75 % of maximal activity from pH 7.5–9.0. Metal ion Mn2+ was required as optimum metal cofactor for activity simulation, but it did not play a significant role in thermostability improvement as reported previously. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for substrate l-arabinose were measured to be 111.68 mM, 773.30/min, and 6.92/mM/min, respectively. The molecular docking results showed that the active site residues of P. haloplanktis L-AI could only immobilize l-arabinose and recognized it as substrate for isomerization.

Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

Lactosucrose, a rare trisaccharide formed from sucrose and lactose by enzymatic transglycosylation, is a type of indigestible carbohydrate with a good prebiotic effect. In this study, lactosucrose biosynthesis was efficiently carried out by a purified levansucrase from Leuconostoc mesenteroides B-512. The target gene was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity by nickel affinity and gel filtration chromatography. The effects of pH, temperature, substrate concentration, substrate ratio, and enzyme amount on lactosucrose biosynthesis were studied in detail, and the optimized conditions were determined to be pH 6.5, 50 °C, 27% (W/V) sucrose, 27% (W/V) lactose, and 5 U mL–1 of the purified recombinant enzyme. Under the optimized reaction conditions, the maximal lactosucrose yield reached 224 g L–1 after reaction for 1 h. Therefore, L. mesenteroides levansucrase could be considered a potential candidate for future industrial production of lactosucrose.

Fructans are the polymers of fructose molecules, normally having a sucrose unit at what would otherwise be the reducing terminus. Inulin and levan are two basic types of simple fructan, which contain β-(2, 1) and β-(2, 6) fructosyl-fructose linkage, respectively. Fructans not only can serve as soluble dietary fibers for food industry, but also may be biologically converted into high-value products, especially high-fructose syrup and fructo-oligosaccharides. In recent years, much attention has been focused on production of difructose dianhydrides (DFAs) from fructans. DFAs are cyclic disaccharides consisting of two fructose units with formation of two reciprocal glycosidic linkages. They are expected to have promising properties and beneficial effects on human health. DFAs can be produced from fructans by fructan fructotransferases. Inulin fructotransferase (IFTase) (DFA III-forming) and IFTase (DFA I-forming) catalyze the DFA III and DFA I production from inulin, respectively, and levan fructotransferase (LFTase) (DFA IV-forming) catalyzes the production of DFA IV from levan. In this article, the DFA-producing microorganisms are summarized, relevant studies on various DFAs-producing enzymes are reviewed, and especially, the comparisons of the enzymes are presented in detail.

Bacterial l-asparaginase catalyzes the hydrolysis of l-asparagine to l-aspartic acid. It is normally used as an antineoplastic drug applied in lymphoblastic leukemia chemotherapy and as a food processing aid in baked or fried food industry to inhibit the formation of acrylamide. The present study demonstrates cloning, expression, and characterization of a thermostable l-asparaginase from Thermococcus zilligii AN1 TziAN1_1 and also evaluates the potential for enzymatic acrylamide mitigation in French fries using this enzyme. The recombinant l-asparaginase was purified to homogeneity by nickel-affinity chromatography. The purified enzyme displayed the maximum activity at pH 8.5 and 90 °C, and the optimum temperature was the highest ever reported. The Km, kcat, and kcat/Km values toward l-asparagine were measured to be 6.08 mM, 3267 s−1, and 537.3 mM−1 s−1, respectively. The enzyme retained 70 % of its original activity after 2 h of incubation at 85 °C. When potato samples were treated with 10 U/mL of l-asparaginase at 80 °C for only 4 min, the acrylamide content in final French fries was reduced by 80.5 % compared with the untreated control. Results of this study revealed that the enzyme was highly active at elevated temperatures, reflecting the potential of the T. zilligiil-asparaginase in the food processing industry.

l-Asparaginases (EC 3.5.1.1) are enzymes that catalyze the hydrolysis of l-asparagine to l-aspartic acid and found in a variety of organisms from microorganisms to mammals. However, they are mainly expressed and produced by microorganisms. Microbial l-asparaginases have received sustained attention due to their irreplaceable role in the therapy of acute lymphoblastic leukemia and for their inhibition of acrylamide formation during food processing. In this article, we review the application of microbial l-asparaginases in medical treatments and acrylamide mitigation. In addition, we describe in detail recent advances in the existing sources, purification, production, properties, molecular modification, and immobilization of l-asparaginase.

A mutation, D478N, was obtained by an error-prone polymerase chain reaction using the l-arabinose isomerase (l-AI) gene from Alicyclobacillus hesperidum URH17-3-68 as the template. The mutated isomerase showed higher activity for d-galactose isomerization. The mutation site obtained from random mutagenesis was then introduced as a single-site mutation using site-directed mutagenesis. Single-site variants, D478N, D478Q, D478A, D478K, and D478R, were constructed. The optimum temperatures were all higher than 60 °C. D478A, D478N, and D478Q retained more than 80 % of the maximum relative activity of the wild-type l-AI at 75 °C. With the exception of the D478A variant, all variants showed decreased optimum pH values in the acidic range (6.0–6.5). All of the variant l-AIs could be significantly activated by the addition of Co2+ and Mn2+. D478N and D478Q showed higher catalytic efficiencies (kcat/Km) toward d-galactose than that of wild-type l-AI. In addition, the D478N and D478Q variants exhibited a much higher conversion ratio of d-galactose to d-tagatose at 6.0 than the wild-type l-AI. According to the molecular model, residue D478 was located on the surface of the enzyme and distant from the active site. It was supposed that the charged state of residue 478 may influence the optimum pH for substrate binding or isomerization.

Monosaccharides are polyhydroxyl compounds containing several chiral carbons, giving rise to tens of isomeric forms. Only minority of them are natural carbohydrates existing in nature abundantly, and most of them are rarely in nature, called rare sugars. These rare sugars attract increasing attention because of their low-calorie property and promising beneficial effects. Izumoring strategy has been established for linking all monosaccharides by three types of enzymes including ketose 3-epimerases, polyol dehydrogenases, and aldose isomerases. Recently, more attention has been paid on the Izumoring of hexoses, especially the D-zone hexoses. So far, at least ten isomerases have been used for the isomerization reactions between D-aldohexoses and D-ketohexoses. In this article, the interconnections and potential physiological effects of D-ketohexoses and D-aldohexoses are summarized, the D-zone hexose Izumoring is shown by giving the exact biocatalysts, and importantly, the isomerases for D-hexose biotransformation are reviewed in detail.

Levansucrase catalyzes three distinct reactions depending on the fructosyl acceptor molecule, including polymerization, transfructosylation, and hydrolysis. As a key biocatalyst in the synthesis of levan and levan-type fructooligosaccharides, levansucrase has been widely and intensively studied. Due to the promising physiological effects of levan and levan-type fructooligosaccharides, they exhibit great potential in food and pharmaceutical industries. Another important point that attracts much attention is the wide substrate specificity of levansucrase toward monosaccharides, disaccharides, and aromatic and alkyl alcohols, producing diverse sucrose analogues, hetero-oligosaccharides (especially lactosucrose), and interesting fructosides. The present article summarizes and discusses the recent applications of microbial levansucrase in detail.

Isomaltulose is a natural isomer of sucrose. It is widely used as a functional sweetener with promising properties, including slower digestion, lower glycemic index, prolonged energy release, lower insulin reaction, and less cariogenicity. It has been approved as a safe sucrose substitute by the Food and Drug Administration of the US; Ministry of Health, Labor and Welfare of Japan; and the Commission of the European Communities. This article presents a review of recent studies on the properties, physiological effects, and food application of isomaltulose. In addition, the biochemical properties of sucrose isomerases producing isomaltulose are compared; the heterologous expression, fermentation optimization, structural determination, and catalysis mechanism of sucrose isomerase are reviewed; and the biotechnological production of isomaltulose from sucrose is summarized.

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s−1, and 2.83 mM−1 s−1, respectively. The Km for NAD+ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.

The rare sugar d-psicose is an ideal sucrose substitute for food products, due to having 70% of the relative sweetness but 0.3% of the energy of sucrose. It also shows important physiological functions. d-Tagatose 3-epimerase (DTEase) family enzymes can produce d-psicose from d-fructose. In this paper, a new member of the DTEase family of enzymes was characterized from Desmospora sp. 8437 (GenBank accession no. WP_009711885) and was named Desmospora sp. d-psicose 3-epimerase (DPEase) due to its highest substrate specificity toward d-psicose. Desmospora sp. DPEase was strictly metal-dependent and displayed maximum activity in the presence of Co2+. The optimum pH and temperature were 7.5 and 60 °C, respectively. The enzyme was relatively thermostable below 50 °C, but easily lost initial activity when preincubated at 60 °C. The thermostability property was almost not affected by the addition of Co2+. Desmospora sp. DPEase had relatively high catalysis efficiency for the substrates d-psicose and d-fructose, which were measured to be 327 and 116 mM–1 min–1, respectively. The equilibrium ratio between d-psicose and d-fructose of Desmospora sp. DPEase was 30:70. The enzyme could produce 142.5 g/L d-psicose from 500 g/L of d-fructose, suggesting that the enzyme is a potential d-psicose producer for industrial production.Keywords: characterization; d-psicose; d-psicose 3-epimerase; Desmospora sp.; rare sugar;

Lactosucrose (O-β-d-galactopyranosyl-(1,4)-O-α-d-glucopyranosyl-(1,2)-β-d-fructofuranoside) is a trisaccharide formed from lactose and sucrose by enzymatic transglycosylation. This rare trisaccharide is a kind of indigestible carbohydrate, has good prebiotic effect, and promotes intestinal mineral absorption. It has been used as a functional ingredient in a range of food products which are approved as foods for specified health uses in Japan. Using lactose and sucrose as substrates, lactosucrose can be produced through transfructosylation by β-fructofuranosidase from Arthrobacter sp. K-1 or a range of levansucrases, or through transgalactosylation by β-galactosidase from Bacillus circulans. This article presented a review of recent studies on the physiological functions of lactosucrose and the biological production from lactose and sucrose by different enzymes.

The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co2+ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the Km, turnover number (kcat), and catalytic efficiency (kcat/Km) for d-psicose were 227 mM, 32,185 min−1, and 141 min−1 mM−1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l−1 was produced from 500 g d-fructose l−1 after 5 h.

Epilactose (4-O-β-d-galactopyranosyl-d-mannose), an epimer of lactose, is a rare disaccharide existing extremely small quantities in heat-treated milk, in which epilactose is produced by non-enzymatic catalysis from lactose. This disaccharide is a kind of non-digestible carbohydrate, has a good prebiotic effect, and promotes intestinal mineral absorption. This article presents a review of recent studies on epilactose formation in food system, qualitative and quantitative analysis, and its physiological functions. In addition, the biochemical properties and kinetic parameters of the epilactose-producing enzyme, cellobiose 2-epimerase, are compared, and the biotechnological production of epilactose from lactose is reviewed.

A cDNA encoding hydroperoxide lyase (HPL) was isolated from Solanum tuberosum, cloned into pQE-30 vector, and expressed in E. coli. The recombinant protein was purified by nickel affinity chromatography and showed an approximate molecular weight of 54 kDa by SDS–PAGE analysis, which was similar to the predicted value based on the putative amino acid sequences (53.9 kDa). 13-Hydroperoxy-linolenic acid (13-HPOT) was the preferred substrate for the enzyme compared with 13-hydroperoxy-linoleic acid (13-HPOD). The corresponding volatile products were 2(E)-hexenal and n-hexanal tested by headspace-gas chromatography, respectively. The enzyme was optimally active at 25 °C and pH 6.5. The Km, Vmax, and the catalytic efficiency (Vmax/Km) for 13-HPOT were 56.6 μM, 71.3 units/mg, and 1.26 units/mg · μM, respectively. Activity of the recombinant potato HPL increased when Triton X-100, sodium chloride, or potassium chloride was added in the reaction mixture, while calcium chloride decreased activity of the recombinant enzyme.

The gene coding for d-lactate dehydrogenase (d-LDH) from Pediococcus acidilactici DSM 20284 was cloned and expressed in E. coli. The recombinant enzyme was purified by nickel-affinity chromatography. It converted phenylpyruvic acid (PPA) to 3-phenyllactic acid maximally at 30°C and pH 5.5 with a specific activity of 140 and 422 U/mg for PPA and pyruvate, respectively. The Km, turnover number (kcat), and catalytic efficiency (kcat/Km) for PPA were 2.9 mM, 305 s−1, and 105 mM−1 s−1, respectively.

3-Phenyllactic acid (PLA), which is an organic acid widely existing in honey and lactic acid bacteria fermented food, can be produced by many microorganisms, especially lactic acid bacteria. It was proved as an ideal antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi. In addition, it could be used as feed additives to replace antibiotics in livestock feeds. This article presented a review of recent studies on the existing resource, antimicrobial activity, and measurement of PLA. In addition, microorganism strains and dehydrogenases producing PLA were reviewed in detail, the metabolic pathway and regulation of PLA synthesis in LAB strains were discussed, and high-level bioproduction of PLA by microorganism fermentation was also summarized.

d-Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of d-fructose and is rarely found in nature. It has 70 % relative sweetness but 0.3 % energy of sucrose, and is suggested as an ideal sucrose substitute for food products. It shows important physiological functions, such as blood glucose suppressive effect, reactive oxygen species scavenging activity, and neuroprotective effect. It also improves the gelling behavior and produces good flavor during food process. This article presents a review of recent studies on the properties, physiological functions, and food application of d-psicose. In addition, the biochemical properties of d-tagatose 3-epimerase family enzymes and the d-psicose-producing enzyme are compared, and the biotechnological production of d-psicose from d-fructose is reviewed.

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co2+. The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co2+, respectively, suggesting that this enzyme was extremely thermostable in the presence of Co2+ but readily inactivated without metal ion. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min–1, and 186.4 mM min–1, respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.

•A psicose-producing DPEase is characterized from Dorea sp. CAG317.•It is the first reported DPEase showing acidic pH optimum (pH 6.0).•It shows significantly high specific activity (803 U/mg).•It exhibits higher productivity of psicose at pH 6.0 than other tested DPEases.Ketose 3-epimerase displayed an important role in not only the cyclic monosaccharides bioconversion strategy, named Izumoring, but also in the industrial biological production of d-psicose, a novel low-calorie rare sugar widely used in food and medical industries. Since the non-enzymatic side reactions could be reduced in acid conditions, slightly acidic pH optimum is one of the main issues for biological production of d-psicose. In this study, we first characterized an acidic ketose 3-epimerase, the recombinant d-psicose 3-epimerase (DPEase) from Dorea sp. CAG317. The protein exhibited high amino acid sequence identity with other reported DPEases, and was determined as a homotetramer with subunit molecular weight approximately 33 kDa, which was the same as other reported findings. The recombinant DPEase was a metal-dependent enzyme with the optimum metal cofactor as Co2+. In presence of 1 mM of Co2+, the enzyme displayed the maximal activity as 803 U/mg at pH 6.0 and 70 °C. The catalytic efficiency (kcat/Km) was measured to be 412 and 199 mM−1 min−1 toward d-psicose and d-fructose, respectively. The equilibrium ratio between d-fructose and d-psicose was approximately 30:70, and the elevated temperature did not significantly shift the equilibrium toward d-psicose. Compared to other reported DPEases, the recombinant Dorea sp. DPEase displayed significantly higher specific activity at acidic pHs and remarkably higher productivity of d-psicose at pH 6.0, indicating that it was appropriate for use as a new source of d-psicose producing enzyme.Download full-size image

•DFA I-producing IFTase was characterized from C. clostridioforme.•The enzyme showed the maximum activity at 50 °C and pH 5.5.•The enzyme was stable under heat treatment of up to 80 °C for 4 h.•The minor products were GF, GF2, GF3, and GF4.•The smallest substrate was GF3.A gene encoding a difructose dianhydride I (DFA I)-forming inulin fructotransferase (IFTase) from Clostridium clostridioforme AGR2157 was cloned and extracellularly expressed in Escherichia coli. SDS-PAGE and gel filtration analysis of the purified enzyme showed molecular mass of 43 and 128 kDa, respectively, indicating that the recombinant enzyme was a trimer with three identical subunits. The enzyme displayed the highest activity at pH 5.5 and 50 °C with the specific activity of 2.076 U mg−1, and it was stable under heat treatment of up to 80 °C for 4 h. In comparison with other reported IFTases (DFA I-forming), the recombinant IFTase from C. clostridioforme demonstrated the best thermostability. Km and Vmax were calculated to be 0.42 mM and 2.69 mM min−1, respectively. The main product of the hydrolysis of inulin by purified enzyme was identified as DFA I according to nuclear magnetic resonance analysis, and the minor products were suggested to be sucrose (GF), 1-kestose (GF2), nystose (GF3), and fructofuranosyl nystose (GF4). The smallest fructo-oligosaccharide substrate was determined to be GF3. When 10, 50, and 100 g L−1 of inulin were catalyzed by the purified enzyme, the maximal yields of DFA I were approximately 85%, 73%, and 71%, respectively.Download full-size image

•T. gammatolerans l-asparaginase showed maximum activity at 85 °C and pH 8.5.•The purified enzyme had specific activities of 7622 U mg−1 for l-Asn.•The enzyme also has catalytic activity toward d-Asn, l-Gln, and d-Gln.•It displayed promising thermostability and a relatively wide pH spectrum.•The Km and kcat/Km for l-Asn were 10.0 mM and 572.1 mM−1 s−1, respectively.Microbial l-asparaginases which catalyze the conversion of l-asparagine to l-asparate and ammonia, have been proved to be useful in medical and food industries. In the present work, a thermostable l-asparaginase was characterized from a hyperthermophilic archaeon strain, Thermococcus gammatolerans EJ3. Cloning and recombinant expression of Tco. gammatolerans l-asparaginase was performed in Escherichia coli. The recombinant enzyme was purified to homogeneity by nickel-affinity chromatography, and was characterized as a homodimer composed of two identical subunits of approximately 36.5 kDa. The optimum pH and temperature were 8.5 and 85 °C, respectively. The purified enzyme had specific activities of 7622 and 2926 U mg−1 for l-asparagine and l-glutamine, respectively, and exhibited promising thermostability at all tested temperatures from 70 to 95 °C. In addition, it displayed very high catalytic efficiency toward substrate l-asparagine. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) values for substrate l-asparagine were estimated to be 10.0 mM, 5721 s−1, and 572.1 mM−1 s−1, respectively.Download full-size image

Rare sugars have recently attracted much attention because of their potential applications in the food, nutraceutical, and pharmaceutical industries. A systematic strategy for enzymatic production of rare sugars, named Izumoring, was developed > 10 years ago. The strategy consists of aldose-ketose isomerization, ketose C-3 epimerization, and monosaccharide oxidation-reduction. Recent development of the Izumoring strategy is reviewed herein, especially the genetic approaches to the improvement of rare sugar-producing enzymes and the applications of target-oriented bioconversion. In addition, novel non-Izumoring enzymatic approaches are also summarized, including enzymatic condensation, phosphorylation-dephosphorylation cascade reaction, aldose epimerization, ulosonic acid decarboxylation, and biosynthesis of rare disaccharides.

•2-CE is characterized from a thermoacidophilic bacterium, T. saccharolyticum.•The enzyme shows maximum activity at 60 °C and pH 7.0.•It produces epilactose as the only product from lactose.•Conversion ratio of lactose to epilactose is 25%.In this study, a recombinant cellobiose 2-epimerase (GenBank accession number, YP_006392930.1) was characterized from a thermophilic bacterium, Thermoanaerobacterium saccharolyticum JW/SL-YS485. The enzyme was metal independent, showed maximal epimerization activity at pH 7.0 and 60 °C, and displayed 29.8, 15.48, and 13.5 U mg−1 for mannobiose, cellobiose, and lactose under optimum conditions, respectively. It exhibited promising thermostability under incubation below 60 °C. The Km, turnover number (kcat), and catalytic efficiency (kcat/Km) for lactose were 124.7 mM, 30.9 s−1, and 0.248 mM−1 s−1, respectively. At pH 7.0 and 60 °C, 50 mM epilactose was produced from 200 mM lactose by a 0.6 μM of enzyme concentration after reaction for 4 h.Download full-size image

•A d-allose-producing L-RI was characterized from T. composti KWC4.•T. composti L-RI showed maximum activity at 65 °C and pH 7.5.•It displayed promising thermostability and a relatively wide pH spectrum.•Tm of T. composti L-RI was increased by 3 °C in presence of Mn2+.•It produced 23.30% d-allose as the sole product from d-allulose.d-Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. l-Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable d-allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn2+ with an optimal pH 7.5 and temperature 65 °C. The Michaelis-Menten constant (Km), turnover number (kcat) and catalytic efficiency (kcat/Km) for l-rhamnose were 33.8 mM, 1189.8 min−1 and 35.2 min−1 mM−1, respectively. At a higher temperature, Mn2+ played a pivotal role in strengthening the thermostability of T. composti L-RI. The differential scanning calorimetry (DSC) results showed the denaturing temperature (Tm) of T. composti L-RI was increased by 3 °C in presence of Mn2+. Although the T. composti L-RI displayed the optimum substrate as l-rhamnose, it could also effectively catalyze the isomerization between d-allulose and d-allose. When the reaction reached equilibrium, the sole product d-allose was produced from D-alluose by T. composti L-RI.Download high-res image (165KB)Download full-size image

3-Phenyllactic acid (PLA) is a broad-spectrum antimicrobial compound, produced by a wide range of lactic acid bacteria. A novel lactic acid bacteria strain with high PLA-producing ability, Pediococcus pentosaceus SK25, was isolated from traditional Chinese pickles. When grown in de Man, Rogosa, Sharpe broth at 30°C for 36 h, this strain produced 135.6 mg/L of PLA. Using this strain as starter for milk fermentation, 47.2 mg/L of PLA was produced after fermentation for 12 h. The PLA production was significantly improved by phenylalanine supplement, but was completely inhibited by tyrosine supplement.

•Purified TnMtDH with high affinity toward fructose to direct production of pure mannitol.•TnMtDH with high catalytic efficiency is proposed as an industrial enzyme.•TnMtDH had unique characters to fulfill the promise of biocatalysis in industrial and medical applications.Mannitol-2-dehydrogenase (MtDH) (E.C. 1.1.1.67) gene was cloned from Thermotoga neapolitana DSM 4359 and expressed in Escherichia coli BL21. The purified enzyme showed a predicted clear band of 36 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native molecular mas was 135 kDa. Km and Vmax values for reduction of D-fructose to D-mannitol were 20 mM and 200 U mg-1 respectively. kcat for reduction direction was 180 s−1 and kcat/Km were 9 mM−1 s−1. The enzyme showed optimal pH at 6.5 and the optimum temperature was 90 °C with 100% relative activity. The purified enzyme was quite stable at 75 °C and had half of initial activity after 1 h of incubation at 90 °C. (TnMtDH) showed no activity with xylitol, inositol, sorbitol, rahmanose, mannose and xylose, and with NADPH and NADP+ as co factors. The presence of some divalent metals in the reaction enhanced the enzyme activity. The enzyme might be utilizing to produce mannitol without other sugar conformation under high temperature.Download high-res image (142KB)Download full-size image

•A thermostable d-lyxose isomerase was characterized from T. oceani.•It displayed maximal activity in presence of 1 mM Mn2+ at pH 6.5 and 65 °C.•The half-life was 5.64, 2.82, 0.77, and 0.2 h at 70, 75, 80, and 85 °C, respectively.d-Mannose has prebiotic effect and potential medical application. Besides, it can be used as substrate to produce mannitol, a functional polyol widely used in food industry. As this result, it has attracted many researchers’ attention. In this work, a thermostable d-mannose-producing d-lyxose isomerase (D-LI) was characterized from a hyperthermophile, Thermosediminibacter oceani. The recombinant D-LI could be remarkably activated by Mn2+. It displayed maximal activity in presence of 1 mM Mn2+ at pH 6.5 and 65 °C, and was determined to be highly thermostable at 80 °C. The half-life was calculated to be 5.64, 2.82, 0.77, and 0.2 h at 70, 75, 80, and 85 °C, respectively. The enzyme showed the optimum activity using d-lyxose as substrate and could also effectively catalyze the isomerization between d-fructose and d-mannose. Under optimum conditions, 101.6 g/L d-mannose was produced from 400 g/L d-fructose after reaction for 9 h, giving a conversion yield of 25.4%.Download full-size image

•l-AI was characterized from a thermoacidophilic bacterium, Al. hesperidum.•The enzyme showed maximum activity at 70 °C and pH 7.0.•It required Co2+ for activity simulation and thermostability improvement.•It displayed promising thermostability and a relatively wide pH spectrum.•The Km and kcat/Km for d-galactose were 54.7 mM and 1.2 mM−1 min−1, respectively.The rare monosaccharide d-tagatose is a low-calorie sugar-substituting sweetener, having 92% of relative sweetness but only 1/3 of energy content of sucrose. Industrial production of d-tagatose is carried out from d-galactose by l-arabinose isomerase (l-AI). It is generally recognized that commercial l-AI for d-tagatose production requires two important enzymatic properties, thermostability and slightly acidic pH optimum. In this article, a thermostable l-AI was characterized from a novel thermoacidophilic bacterium, Alicyclobacillus hesperidum URH17-3-68, which showed promising thermostability and displayed a relatively wide pH spectrum. The araA gene encoding the Al. hesperidum l-AI was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and ion-exchange chromatography. The enzyme displayed maximal activity at 70 °C and pH 7.0, and showed more than 75% of maximal activity from pH 5.5 to 7.0. Cobalt ion was required as optimum metal cofactor for both activity simulation and thermostability improvement at high temperature. The enzyme retained 93% and 63% of initial activity after 4 and 16 h of incubation, respectively, at 75 °C in the presence of Co2+. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for substrate d-galactose were measured to be 54.7 mM, 68.0 min−1, and 1.2 mM−1 min−1, respectively. The bioconversion yield of d-tagatose by the purified enzyme after 27 h at 70 °C reached 43% and 22%, from 50 and 200 mM of d-galactose, respectively. Due to the promising thermostability and high activity at slight acidic pH, the Al. hesperidum l-AI was appropriate for use as a new source of d-tagatose producing enzyme.Download full-size image