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D-tagatose, a low-calorie rare sugar, has significant potential in food, medicine, cosmetics, and other industries owing to its high application value and market potential. In this study, Escherichia coli BL21 was used as the starting strain to express the β-galactosidase (β-Gal) gene—BgaB—derived from Bacillus stearothermophilus and the L-arabinose isomerase (L-AI) gene—araA—derived from Thermus sp., yielding the genetically engineered strains E. coli BL21-pET28a-BgaB and E. coli BL21-pET28a-araA. These strains synthesized D-tagatose using β-Gal and L-AI with a conversion rate of 23.73%. Based on this, we constructed a multienzyme cascade pathway comprising β-Gal, L-AI, glucose isomerase (GI), fructose kinase (FK), D-tagatose-bisphosphate aldolase (GatZ), polyphosphate kinase (PPK), and phosphatase (PGP), further enhancing D-tagatose biosynthesis. This multienzyme approach improved the conversion of the intermediate product D-glucose to D-tagatose by 3.84% compared with the dual-enzyme system. Thus, our study provides a theoretical basis and technical support for the industrial production of D-tagatose. [ABSTRACT FROM AUTHOR]

D-tagatose is a valuable rare sugar with potential health benefits such as antiobesity, low-calorie, prebiotic, and anticancer. However, its production is mainly depending on chemical or enzymatic catalysis. Herein, a cobalt-based metal–organic framework (MOF) was developed at room temperature in an aqueous system using a self-assembly method. The L-arabinose isomerase (L-AI) was immobilized into this unique MOF by an in situ encapsulation process. The morphology and structural aspects of the MOF preparations were characterized by different analytical techniques such as scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), confocal laser scanning microscopy (CLSM), Fourier transform infrared spectroscopy (FT-IR), and X-Ray diffraction (XRD). Moreover, thermogravimetric analysis (TGA) suggested the high thermal stability of the L-AI@MOF. Significantly, the immobilized catalyst exhibited enhanced catalytic efficiency (kcat/Km) of 3.22 mM−1 s−1 and improved turnover number (kcat) of 57.32 s−1. The L-AI@MOF efficiently catalyzes the synthesis of D-tagatose from D-galactose up to the equilibrium level (~ 50%) of isomerization in heterogeneous catalysis. Interestingly, L-AI@MOF was found stable and reusable for more than five cycles without the requirement of additional metal ions during catalysis. Thus, L-AI stabilized in the MOF system demonstrated a higher catalytic activity and potential guidance for the sustainable synthesis of rare sugar D-tagatose. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Dalian Polytechnic University is the property of Journal of Dalian Polytechnic University Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of Lactobacillus plantarum CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Dalian Polytechnic University is the property of Journal of Dalian Polytechnic University Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

D-Tagatose is a low-calorie sugar substitute that has gained increased attention as a functional sweetener owing to its nutraceutical and prebiotic properties. Traditionally, D-tagatose is produced via the enzymatic conversion of L-galactose to D-tagatose by L-arabinose isomerase (L-AI). Nonetheless, the most reported L-AI enzymes are ion-dependent enzymes requiring Mn2+ and/or Co2+ as cofactors for their reactions, which limits their application due to safety and health concerns. Herein, we addressed the facile bioconversion of L-galactose to D-tagatose using a novel recombinant metallic-ions-independent L-AI derived from endophytic Bacillus amyloliquefaciens CAAI isolated from cantaloupe fruits. The ORF (1500 bp) of the L-arabinose isomerase gene (araA) was cloned and over-expressed in Escherichia coli. The recombinant enzyme (BAAI) was purified to homogeneity using Ni-NTA affinity chromatography, yielding a single distinct band with an apparent molecular mass of approximately 59 kDa as deduced from SDS-PAGE analysis. The purified enzyme showed optimum activity at pH and temperature of 7.5 and 45 °C, respectively, with obvious enzymatic activity in the presence of ethylenediaminetetraacetic acid (EDTA), indicating the metallic-ions independence from BAAI. The Km values of BAAI for D-galactose and L-arabinose were 251.6 mM and 92.8 mM, respectively. The catalytic efficiency (kcat/Km) values for D-galactose and L-arabinose were found to be 2.34 and 46.85 mM–1 min–1, respectively. The results revealed the production of 47.2 g/L D-tagatose from D-galactose (100 g/L) with 47.2% bioconversion efficiency in a metallic-ions-free reaction system that could be implemented in safe-production of food-grade low-calorie sweetener, D-tagatose. [ABSTRACT FROM AUTHOR]

L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of Lactobacillus plantarum CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose.

Rare sugar was defined as a sugar that occurs in very small quantities in nature. Among them, l -ribose and d -tagatose were of high added value and useful as pharmaceutical intermediate for anti-HBV drugs or low calorie sweetener in food industry. Bio-production of the two rare sugar from biomass waste has not been investigated. Hence, development of a feasible and efficient co-production method was of practical usage. However, lack of suitable biocatalyst has become a bottleneck. By sequence alignment and analysis, a C-terminal α-helix from l -arabinose isomerase (L-AI) family was selected as a tool for protein engineering. This α-helix was ligated to C-terminal of Lactobacillus fermentum L-AI (LFAI) and significantly enhanced its thermostability and robustness for both l -arabinose and galactose catalysis. The mutant LFAI-C4 enzyme was immobilized by alginate and antimicrobial peptide poly- l -lysine, and was used to convert pretreated corncob acid hydrolysate (PCAH) into l -ribulose and d -tagatose in the presence of boric acid. In addition, we identified and immobilized a novel thermostable mannose-6-phosphate isomerase from Bacillus subtilis (BsMPI-2) which was efficient in catalyzing retaining l -ribulose into l -ribose and showing no activity on d -tagatose. The dual immobilized enzymes (LFAI-C4 and BsMPI-2) system co-produced 191.9 g/L l -ribose and 80.1 g/L d -tagatose, respectively. Showing a total yield of 46.6% from l -arabinose to l -ribose, which was the highest among reported. The dual immobilized enzymes system preserved 82% activity after 40 batches reaction, showing excellent potentials for industrial use. This study presents a promising alternative for rare sugar production from low-value raw material and showed satisfied conversion rate, product concentration, and operation stability. • An α-helix ligated to l -arabinose isomerase significantly enhanced thermostability and robustness. • The modified L-AI was used to convert pretreated corncob acid hydrolysate into l -ribulose and d -tagatose in high efficiency. • A novel thermostable mannose-6-phosphate isomerase from Bacillus subtilis was identified. • The dual L-AI and MPI immobilized enzymes system co-produced high concentrations of rare sugar. • The dual immobilized enzymes system preserved most activity after long term bio-reaction. [ABSTRACT FROM AUTHOR]

D-Tagatose is a low-calorie sugar substitute that has gained increased attention as a functional sweetener owing to its nutraceutical and prebiotic properties. Traditionally, D-tagatose is produced via the enzymatic conversion of L-galactose to D-tagatose by L-arabinose isomerase (L-AI). Nonetheless, the most reported L-AI enzymes are ion-dependent enzymes requiring Mn2+ and/or Co2+ as cofactors for their reactions, which limits their application due to safety and health concerns. Herein, we addressed the facile bioconversion of L-galactose to D-tagatose using a novel recombinant metallic-ions-independent L-AI derived from endophytic Bacillus amyloliquefaciens CAAI isolated from cantaloupe fruits. The ORF (1500 bp) of the L-arabinose isomerase gene (araA) was cloned and over-expressed in Escherichia coli. The recombinant enzyme (BAAI) was purified to homogeneity using Ni-NTA affinity chromatography, yielding a single distinct band with an apparent molecular mass of approximately 59 kDa as deduced from SDS-PAGE analysis. The purified enzyme showed optimum activity at pH and temperature of 7.5 and 45 °C, respectively, with obvious enzymatic activity in the presence of ethylenediaminetetraacetic acid (EDTA), indicating the metallic-ions independence from BAAI. The Km values of BAAI for D-galactose and L-arabinose were 251.6 mM and 92.8 mM, respectively. The catalytic efficiency (kcat/Km) values for D-galactose and L-arabinose were found to be 2.34 and 46.85 mM–1 min–1, respectively. The results revealed the production of 47.2 g/L D-tagatose from D-galactose (100 g/L) with 47.2% bioconversion efficiency in a metallic-ions-free reaction system that could be implemented in safe-production of food-grade low-calorie sweetener, D-tagatose.

L-arabinose isomerase (L-AI) is a functional enzyme for the isomerizing of D-galactose to produce D-tagatose. In this study, L-AI-C6-encoding gene from the probiotic Lactobacillus fermentum C6 was cloned and expressed in Bacillus subtilis WB600 for investigating enzymatic characteristics and bioconverting D-tagatose by means of whole-cell catalysis. Results showed that the engineered B. subtilis WB600-pMA5-LAI achieved a maximum specific activity of L-AI-C6 (232.65 ± 15.54 U/mg protein) under cultivation in LB medium at 28 °C for 40 h. The recombinant L-AI-C6 was purified, and enzymatic characteristics test showed its optimum reaction temperature and pH at 60 °C and 8.0, respectively. In addition, L-AI-C6 exhibited good stability within the pH range of 5.5–9.0. By using B. subtilis WB600-pMA5-LAI cells as whole-cell catalyst, the highest D-tagatose yield reached 42.91 ± 0.28 % with D-galactose as substrate, which was 2.41 times that of L. fermentum C6 (17.79 ± 0.11 %). This suggested that the cloning and heterologous expression of L-AI-C6 was an effective strategy for improving D-tagatose conversion by whole-cell catalysis. In brief, the present study demonstrated that the reaction temperature, pH, and stability of L-AI-C6 from L. fermentum C6 meet the demands of industrial application, and the constructed B. subtilis WB600-pMA5-LAI shows promising potential for the whole-cell biotransformation of D-tagatose. [Display omitted] • L-AI-C6 originates from the GRAS Lactobacillus fermentum C6. • L-AI-C6 exhibits excellent enzymatic properties for industrial application. • The engineered B. subtilis shows promising whole-cell catalytic activity on D-tagatose production. [ABSTRACT FROM AUTHOR]

D-Galactose is an abundant carbohydrate monomer in nature and widely exists in macroalgae, plants, and dairy wastes. D-Galactose is useful as a raw material for biomass fuel production or low-calorie sweetener production, attracting increased attention. This article summarizes the studies on biotechnological processes for galactose utilization. Two main research directions of microbial fermentation and enzyme-catalyzed conversion from galactose-rich biomass are extensively reviewed. The review provides the recent discoveries for biofuel production from macroalgae, including the innovative methods in the pretreatment process and technological development in the fermentation process. As modern people pay more attention to health, enzyme technologies for low-calorie sweetener production are more urgently needed. D-Tagatose is a promising low-calorie alternative to sugar. We discuss the recent studies on characterization and genetic modification of L-arabinose isomerase to improve the bioconversion of D-galactose to D-tagatose. In addition, the trends and critical challenges in both research directions are outlined at the end. Key points: • The value and significance of galactose utilization are highlighted. • Biofuel production from galactose-rich biomass is accomplished by fermentation. • L-arabinose isomerase is a tool for bioconversion of D-galactose to D-tagatose. [ABSTRACT FROM AUTHOR]

Abstract d-Mannose and l-ribose are two important monosaccharides, which have attracted public attention recently because of their great application potentials in food, cosmetic and pharmaceutical industries. Sugar isomerases catalyze the sugar isomerization and therefore can be used as the biocatalysts for production of the high-value sugars from inexpensive sugars. l-arabinose isomerase catalyzes the conversion of l-arabinose to l-ribulose, while d-lyxose isomerase catalyzes l-ribulose and d-fructose to l-ribose and d-mannose, respectively. In this paper, a putative d-LI from Bacillus velezensis (BvLI) was identified, characterized and used to produce d-mannose and l-ribose from d-fructose and l-arabinose, respectively. The recombinant BvLI exhibited a maximum activity at 55 °C and pH 6.5, in the presence of 0.1 mM Co2+. Approximately 110.75 g/L d-mannose was obtained from 500 g/L d-fructose in 6 h by the recombinant BvLI, and approximately 105 g/L l-ribose was obtained from 500 g/L l-arabinose in 8 h by the successive biocatalysis of l-arabinose isomerase from Bacillus licheniformis (BlAI) and BvLI.

Copyright of Journal of Dalian Polytechnic University is the property of Journal of Dalian Polytechnic University Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This study reports an alternative strategy for the expression of a recombinant l-AI from Enterococcus faecium DBFIQ E36 by auto-induction using glucose and glycerol as carbon sources and residual whey lactose as inducer agent. Commercial lactose and isopropyl β-d-1-thiogalactopyranoside (IPTG) were also evaluated as inducers for comparison of enzyme expression levels. The enzymatic extracts were purified by affinity chromatography, characterized, and applied in the bioconversion of d-galactose into d-tagatose. l-AI presented a catalytic activity of 1.67 ± 0.14, 1.52 ± 0.01, and 0.7 ± 0.04 U/mL, when expressed using commercial lactose, lactose from whey, and IPTG, respectively. Higher activities could be obtained by changing the protocol of enzyme extraction and, for instance, the enzymatic extract produced with whey presented a catalytic activity of 3.8 U/mL. The specific activity of the enzyme extracts produced using lactose (commercial or residual whey) after enzyme purification was also higher when compared to the enzyme expressed with IPTG. Best results were achieved when enzyme expression was conducted using 4 g/L of residual whey lactose for 11 h. These results proved the efficacy of an alternative and economic protocol for the effective expression of a recombinant l-AI aiming its high-scale production. [ABSTRACT FROM AUTHOR]

D-Galactose-specific L-arabinose isomerase (L-AI) would have much potential for the enzymatic conversion of D-Galactose into D-tagatose, while most of the reported L-AIs are L-arabinose specific. This study explored a highly D-Galactose-specific L-AI from Bifidobacterium adolescentis (BAAI) for the production of D-tagatose. In the comparative protein-substrate docking for D-Galactose and L-arabinose, BAAI showed higher numbers of hydrogen bonds in D-Galactose-BAAI bonding site than those found in L-arabinose-BAAI bonding site. The activity of BAAI was 24.47 U/mg, and it showed good stability at temperatures up to 65°C and a pH range 6.0–7.5. The Km, Vmax, and Kcat/Km of BAAI were found to be 22.4 mM, 489 U/mg and 9.3 mM–1 min–1, respectively for D-Galactose, while the respective values for L-arabinose were 40.2 mM, 275.1 U/mg, and 8.6 mM–1 min–1. Enzymatic conversion of D-Galactose into D-tagatose by BAAI showed 56.7% conversion efficiency at 55°C and pH 6.5 after 10 h.

d-Tagatose, a functional sweetener, is converted from d-galactose by l-arabinose isomerase, which catalyzes the conversion of l-arabinose to l-ribulose. In this study, the araA gene encoding l-arabinose isomerase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli, and the expressed enzyme was purified and characterized. The purified l-arabinose isomerase, a soluble protein with 11.6-fold purification and a 22% final yield, displayed a specific activity of 1.8 U/mg for d-galactose and existed as a homohexamer of 336 kDa. The enzyme exhibited maximum activity at pH 8.0 and 40 °C in the presence of Mn2+ and relative activity for pentoses and hexoses in the order l-arabinose > d-galactose > l-ribulose > d-xylulose > d-xylose > d-tagatose > d-glucose. The thermal stability of recombinant E. coli cells expressing l-arabinose isomerase from K. pneumoniae was higher than that of the enzyme. Thus, the reaction conditions of the recombinant cells were optimized to pH 8.0, 50 °C, and 4 g/L cell concentration using 100 g/L d-galactose with 1 mM Mn2+. Under these conditions, 33.5 g/L d-tagatose was produced from d-galactose with 33.5% molar yield and 67 g/L/h productivity. Our findings will help produce d-tagatose using whole-cell reactions, extending its industrial application.

As a substitute sweetener for sucrose, d‐tagatose is widely used in products, such as health drinks, yogurt, fruit juices, baked goods, confectionery, and pharmaceutical preparations. In the fermentation process of l‐AI produced by Lactobacillus plantarum, d‐tagatose is produced through biotransformation and this study was based on the fermentation process of Lactobacillus plantarum WU14 producing l‐AI to further research the biotransformation and separation process of d‐tagatose. The kinetics of cell growth, substrate consumption, and l‐arabinose isomerase formation were established by nonlinear fitting, and the fitting degrees were 0.996, 0.994, and 0.991, respectively, which could better reflect the change rule of d‐tagatose biotransformation in the fermentation process of L. plantarum WU14. The separation process of d‐tagatose was identified by decolorization, protein removal, desalination, and freeze drying, initially. Finally, the volume ratio of whole cell catalysts, d‐galactose, and borate was 5:1:2 at 60°C, pH 7.17 through borate complexation; then, after 24 hr of conversion, the yield of d‐tagatose was 58 g/L. [ABSTRACT FROM AUTHOR]

d -tagatose is regarded as one of the most valuable rare sugars in the current market and its biotechnological ways of production is a very hot topic as of today. As a ketohexose, d -tagatose having low-caloric value and 90% of sweetness with sucrose is a rare monosaccharide that exists extremely in low quantities in nature. Owing to distinct properties and beneficial functions on human health it has received a great interest and attained Generally Recognised as Safe (GRAS) status by the U.S. Food and Drug Administration (USFDA) in 2010. Due to scarcity in nature, production of d -tagatose either by chemical or biological methods is of utmost importance. In order to overcome the challenges that occur in chemical production, recently, biological process by using l -arabinose isomerase (L -AIs) for converting d -galactose to d -tagatose has allured researchers to find out more new ways to synthesize it in a cheap and eco-friendly manner. Herein, a brief summary on the scope of enzyme engineering methods like rational, semi-rational and directed evolution approaches to generate tailored L -AIs for improving d -tagatose yield and its function under harsh conditions are summarized. In addition, comparison of biochemical parameters and structural key aspects are also highlighted in this paper. Till date, L -AIs from various microbial sources are identified and characterized. Elucidating crystal structures and catalytic mechanism has elevated the researcher's interest to modify the L -AIs at molecular level and demonstrate its potential that is suited for industrial settings. However, works to produce d -tagatose from cheap renewable feedstocks and methods like immobilization and cell-surface display of L -AIs for efficient uptake and biotransformation of d -galactose are scarcely reported. In future, by employing the aforementioned strategies, the research studies must be focused more on shaping L -AIs as powerful biocatalysts to produce d -tagatose at industrial scale that necessitates for the human welfare. • Scope and importance of l -arabinose isomerase (L -AI) for biological production of d -tagatose is reviewed. • Engineering L -AI by various protein engineering methods is comprehensively discussed. • Feasibility of using renewable sources and industrial by-products wastes for d -tagatose synthesis are highlighted. • Importance of immobilization and other cellular engineering for enhancing d -tagatose yield or efficient substrate utilization are discussed. • Use of unnatural amino acid mutagenesis and downstream processing research needs are highlighted in future research area. [ABSTRACT FROM AUTHOR]

Copyright of Iraq Journal of Market Research & Consumer Protection / Al-Mağallaẗ al-ʿIrāqiyyaẗ li-Buḥūṯ al-Sūq wa-Ḥimāyaẗ al-Mustahlik is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A recombinant l-arabinose isomerase from Enterococcus faecium DBFIQ E36 was immobilized onto multifunctional epoxide supports by chemical adsorption and onto a chelate-activated support via polyhistidine-tag, located on the N-terminal (N-His-L-AI) or on the C-terminal (C-His-L-AI) sequence, followed by covalent bonding between the enzyme and the support. The results were compared to reversible L-AI immobilization by adsorption onto charged agarose supports with improved stability. All the derivatives presented immobilization yields of above 75%. The ionic interaction established between agarose gels containing monoaminoethyl-N-aminoethyl structures (MANAE) and the enzyme was the most suitable strategy for L-AI immobilization in comparison to the chelate-activated agarose. In addition, the immobilized biocatalysts by ionic interaction in MANAE showed to be the most stable, retaining up to 100% of enzyme activity for 60 min at 60 °C and with Km values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively. [ABSTRACT FROM AUTHOR]

d-Tagatose is a ketohexose, which presents unique properties as a low-calorie functional sweetener possessing a sweet flavor profile similar to d-sucrose and having no aftertaste. Considered a generally recognized as safe (GRAS) substance by FAO/WHO, d-tagatose can be used as an intermediate for the synthesis of other optically active compounds as well as an additive in detergent, cosmetic, and pharmaceutical formulations. This study reports important features for l-arabinose isomerase (EC 5.3.1.4) (L-AI) use in industry. We describe arabinose (araA) gene virulence analysis, gene isolation, sequencing, cloning, and heterologous overexpression of L-AI from the food-grade GRAS bacterium Enterococcus faecium DBFIQ E36 in Escherichia coli and assess biochemical properties of this recombinant enzyme. Recombinant L-AI (rL-AI) was one-step purified to homogeneity by Ni2+-agarose resin affinity chromatography and biochemical characterization revealed low identity with both thermophilic and mesophilic L-AIs but high degree of conservation in residues involved in substrate recognition. Optimal conditions for rL-AI activity were 50 °C, pH 5.5, and 0.3 mM Mn2+, exhibiting a low cofactor concentration requirement and an acidic optimum pH. Half-life at 45 °C and 50 °C were 1427 h and 11 h, respectively, and 21.5 h and 39.5 h at pH 4.5 and 5.6, respectively, showing the high stability of the enzyme in the presence of a metallic cofactor. Bioconversion yield for d-tagatose biosynthesis was 45% at 50 °C after 48 h. These properties highlight the technological potential of E. faecium rL-AI as biocatalyst for d-tagatose production. [ABSTRACT FROM AUTHOR]

Fifteen of local isolates of Bacillus sp. were obtained among forty isolates from different location of Iraqi soil. These isolate were subjected to primary and secondary screening and their ability for L-arabinose isomerase production were tested. The isolate A3 was the the highest in enzyme production with enzyme activity 30.53 unit/ml. The morphological and biochemical tests for isolate A3 revealed that this isolate belong to Bacillus subtilis and signed as AH1. Crude enzyme of L-arabinose isomerasefrom Bacillus subtilis AH1 was produced under optimum condition by submerged culture and partially purified by two steps including, concentration with 60% saturation of ammonium sulfate and dialysis. The results revealed that the purification folds was 1.54 and yield were 24.20%. The optimium pH for enzyme activity were 7.5 and the optimum pH of stability between 7-8, while the optimum temperature for enzyme activity was 50°C and the optimum temperature for stability between30 -50oC for 30 min of incubation. [ABSTRACT FROM AUTHOR]

The one-pot production of tagatose from lactose using recombinant l -arabinose isomerase (L-AI) from Thermotoga maritima and commercial β-galactosidase (β-gal) from Aspergillus oryzae was evaluated. The β-gal and L-AI ratio (R β-gal/L-AI), initial lactose concentration and temperature were chosen as study variables. Maximum values of tagatose yield (Y Tag), specific tagatose productivity (π Tag) and sugars ratio (R Tag/Gal) obtained were 23 %, 0.202 ± 0.03 mmol Tag/(g prot⋅h) and 1, respectively. The composition of the product on a dry basis was as follows (g/g): 0.23 tagatose, 0.232 galactose, 0.454 glucose, 0.043 fructose, 0.029 lactose and 0.012 GOS. The one-pot production was superior compared with a sequential operation, where values of 16.5 %, 0.11 mmol Tag/(g prot⋅h) and 0.59 were obtained for Y Tag , π Tag and R Tag/Gal , respectively. • Tagatose was produced in one-pot operation with A. oryzae βgal and T. maritima L-AI. • Higher yield and specific productivity were achieved in one-pot operation. • Thermodynamically and kinetically controlled reaction are involved in one-pot operation. [ABSTRACT FROM AUTHOR]

A novel l -arabinose isomerase (L-AI) from Arthrobacter psychrolactophilus (Ap L-AI) was successfully cloned and characterized. The enzyme catalyzes the isomerization of d -galactose into a rare sugar d -tagatose. The recombinant Ap L-AI had an approximate molecular weight of about 258 kDa, suggesting it was an aggregate of five 58 kDa monomers and became the first record as a homo-pentamer L-AI. The catalytic efficiency (k cat /Km) and Km for d -galactose were 0.32 mM−1 min−1 and 51.43 mM, respectively, while for l -arabinose, were 0.64 mM−1 min−1 and 23.41 mM, respectively. It had the highest activity at pH 7.0–7.5 and 60 °C in the presence of 0.250 mM Mn2+. Ap L-AI was discovered to be an outstanding thermostable enzyme that only lost its half-life value at 60 °C for >1000 min. These findings suggest that l -arabinose isomerase from Arthrobacter psychrolactophilus is a promising candidate for d -tagatose mass-production due to its industrially competitive temperature. • Sequence of L-AI retrieved from the genome of Arthrobacter psychrolactophilus B7 • Gene cloning and overexpression in E. coli host using pET-28a(+) • Enzyme activity characterization indicated high thermal stability. [ABSTRACT FROM AUTHOR]

L-arabinose isomerase (L-AI) (EC 5. 3. 1. 4. L-AI) that mediates the isomerization of D-galactose to D-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1 kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The L-AI was found to be stable at 45-75 °C, and at pH 7.0-9.0. Its optimum temperature and pH was determined as 65 °C and 7.0, respectively. Besides, we found that Ca2+, Cu2+, and Ba2+ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg2+, Mn2+, or Co2+ ions. The optimum concentration of Mn2+ and Co2+ was determined to be 1 mM. Furthermore, we characterized the kinetic parameters for L-AI and determined the Km (129 mM) and the Vmax (0.045 mM min− 1) values. Notably, L. brevisL-AI exhibited a high bioconversion yield of 43% from D-galactose to D-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other L-AIs from various organisms. [ABSTRACT FROM AUTHOR]

Abstract L-Arabinose isomerase isolated from Geobacillus stearothermophilus (GSAI) was modified to improve its substrate specificity for D-galactose for the production of D-tagatose, a potential reduced-energy sweetener. Among the selected residues, mutation at residue 18 produced a mutant strain, H18T, which exhibited increased activity for D-galactose compared with the wild-type (WT) enzyme. Analysis of the substrate specificity of H18T showed a 45.4% improvement for D-galactose. Replacing histidine with threonine at residue 18 resulted in approximately 2.7-fold and 1.8-fold higher substrate binding and catalytic efficiency, respectively, for D-galactose. Further enhancement of the specific activity and catalytic efficiency of H18T for D-galactose by up to 2.7-fold and 4.3-fold, respectively, was achieved by adding borate during L-arabinose isomerase catalysis. Moreover, H18T showed thermostability and no destabilization was detected, which is promising for the industrial production of D-tagatose. Highlights • Mutation from histidine to threonine at position 18 (H18T) of GSAI improved the substrate specificity for D-galactose. • The H18T exhibited no destabilization after mutation. • H18T possessed flexibility and fluctuation around the binding pocket compared to that of WT. • The addition of borate further facilitated the conversion toward D-tagatose. [ABSTRACT FROM AUTHOR]

Nutraceuticals are gaining importance owing to their potential applications in numerous sectors including food and feed industries. Among the emerging nutraceuticals, d‐tagatose occupies a significant niche because of its low calorific value, antidiabetic property and growth promoting effects on beneficial gut bacteria. As d‐tagatose is present in minute quantities in naturally occurring food substances, it is produced mainly by chemical or biological means. Recently, attempts were made for bio‐production of d‐tagatose using l‐arabinose isomerase enzyme to overcome the challenges of chemical process of production. Applications of d‐tagatose for maintaining health and wellbeing are increasing due to growing consumer awareness and apprehension against modern therapeutic agents. This review outlines the current status on d‐tagatose, particularly its production, properties, biological role, applications, and the future perspectives. [ABSTRACT FROM AUTHOR]

L-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg-1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve. [ABSTRACT FROM AUTHOR]

D-tagatose is a functional sweetener present in medicine, food, and dairy products and with broad market prospects, and L-arabinose isomerase gene (araA) can mediate the bioconversion of D-galactose into D-tagatose. In this study, a Lactococcus lactis NZ9000 strain harboring exogenous L-arabinose isomerase was constructed to produce D-tagatose. Lactobacillus plantarum CGMCC 8198 exhibits L-arabinose isomerase activity and its genome has been sequenced. The araA gene of Lactobacillus plantarum CGMCC 8198 encoding L-arabinose isomerase was identified by sequence analysis and was successfully cloned and expressed in Lactococcus lactis NZ9000. The D-tagatose production by the whole cell of the recombinant strain was optimized. The optimal condition for conversion reaction was at 50°C, pH 7.0 and with 300 mmol/L Mn2+ and 60 g/L galactose added. The D-tagatose yield and conversion rate at the optimal condition was determined and reached 40.2 g/L and 67%, respectively. [ABSTRACT FROM AUTHOR]

d -Tagatose is a novel low-calorie sweetener, but its application in milk and yogurt has not been explored. A novel l -arabinose isomerase gene was identified in Lactobacillus rhamnosus. The purified l -arabinose isomerase showed good thermal stability from 50 °C to 65 °C, and pH stability from 6.0 to 8.0. To prepare sweet milk, 3.77 × 10−7 kat/mL β-galactosidase and 9.02 × 10−8 kat/mL l -arabinose isomerase were added. After reaction at 65 °C for 3.5 h, the contents of glucose, d -galactose, and d -tagatose in milk were 25.00 g/L, 13.76 g/L, and 11.24 g/L, respectively, and lactose was not detected. Furthermore, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus , with the respective characteristic of d -galactose, and glucose utilization were used as starters for sweet yogurt preparation. After fermentation, the yogurt contained 13.88 g/L of glucose, 10.13 g/L of d -galactose, and 10.90 g/L of d -tagatose, respectively. These results indicated the L. rhamnosus l -arabinose isomerase had a great potential for industrial application in sweetened milk and yogurt preparation. • A l -arabinose isomerase derived from L. rhamnosus was expressed in E. coli. • The enzyme had a high optimum temperature of 65 °C and good thermal stability. • Combined action of l -arabinose isomerase and β-galactosidase was explored. • The sweet milk and yogurt containing d -tagatose were prepared by the enzymes. [ABSTRACT FROM AUTHOR]

The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, β-galactosidase from Bacillus circulans, L-arabinose (D-galactose) isomerase from Enterococcus faecium, and D-xylose (D-glucose) isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing L-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and D-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system. [ABSTRACT FROM AUTHOR]

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 Mn 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 ( K ), turnover number ( k ), and catalytic efficiency ( k / K ) 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. [ABSTRACT FROM AUTHOR]

Background: L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed. Results: The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn2+ at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (kcat/Km) for L-arabinose and D-galactose were 8.7 mM-1 min-1 and 1.0 mM-1 min-1 respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L-1 and 250 g L-1 D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h). Conclusions: In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn2+. Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system. [ABSTRACT FROM AUTHOR]

Aim of present study was to develop biological catalysts of L-arabinose isomerase (L-AI) by immobilizing on four different supports such as multiwalled carbon nanotube (MWCNT), graphene oxide (GOx), Santa Barbara Amorphous (SBA-15) and mobile composite matter (MCM-41). Also, comparative analysis of the developed catalysts was performed to evolve the best in terms of transformation efficiency for D-tagatose production. The developed nano-enzyme conjugates (NECs) were characterized using the high resolution transmission electron microscopy (HR-TEM) and elemental analysis was performed by energy dispersive X-ray spectroscopy (EDS). The functional groups were investigated by Fourier transform infra red spectroscopy. Also, the thermo gravimetric analysis (TGA) was employed to plot a thermal degradation weight loss profile of NECs. The conjugated L-AI with MWCNT and GOx were found to be more promising immobilized catalysts due to their ability to provide more surface area. Conversion of D-Galactose to D-Tagatose at moderate temperature and pH was observed to attain the equilibrium level of transformation (~50%). On the contrary, NECs prepared using SBA-15 and MCM-41 as support matrix were unable to reach the equilibrium level of conversion. Additionally, the developed NECs were suitable for reuse in multiple batch cycles. Thus, promising nanotechnology coupled with biocatalysis made the transformation of D-Galactose into D-tagatose more economically sustainable. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The l-arabinose isomerase is an intracellular enzyme which converts l-arabinose into l-ribulose in living systems and d-galactose into d-tagatose in industrial processes and at industrial scales. d-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The d-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive l-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of d-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. Results: The l-arabinose isomerase (l-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SPUsp45). The l-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei l-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant l-AI with the SPUsp45. Th l-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the l-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. Conclusions: We report for the first time the secretion of the intracellular l-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted l-AI originated from the food grade L. sakei 23 K was active and showed the same catalytic and structural properties as the intracellular enzyme. The L. lactis strains secreting the l-arabinose isomerase has the ability to produce d-tagatose in vivo and conferred an antihyperglycemic effect to mice. [ABSTRACT FROM AUTHOR]

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 Co and Mn. D478N and D478Q showed higher catalytic efficiencies ( k/ K) 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. [ABSTRACT FROM AUTHOR]

d-tagatose is produced from d-galactose by the enzyme l-arabinose isomerase (L-AI) in a commercially viable bioprocess. An active and stable biocatalyst was obtained by modifying chitosan gel structure through reaction with TNBS, d-fructose or DMF, among others. This led to a significant improvement in L-AI immobilization via multipoint covalent attachment approach. Synthetized derivatives were compared with commercial supports such as Eupergit C250L and glyoxal-agarose. The best chitosan derivative for L-AI immobilization was achieved by reacting 4 % (w/v) d-fructose with 3 % (w/v) chitosan at 50 °C for 4 h. When compared to the free enzyme, the glutaraldehyde-activated chitosan biocatalyst showed an apparent activity of 88.4 U g with a 211-fold stabilization factor while the glyoxal-agarose biocatalyst gave an apparent activity of 161.8 U g with an 85-fold stabilization factor. Hence, chitosan derivatives were comparable to commercial resins, thus becoming a viable low-cost strategy to obtain high active L-AI insolubilized derivatives. [ABSTRACT FROM AUTHOR]

L-Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was overexpressed in Escherichia coli as a (His)6-tagged protein and purified by heat treatment followed by immobilized Ni2+ affinity chromatography. TMAI from a heat-treated preparation was immobilized on the epoxy support Eupergit C250L (Eu) and on its copper–chelate form (Eu–Cu). The immobilization yield and the specific activity at 80 °C and pH 7.5 for the former or the latter derivatives were, respectively, 7.2 or 25 mg BSAE per gds (grams of dry solid) and 0.44 ± 0.04 or 3.1 ± 0.4 IU per gds. TMAI immobilized on Eu–Cu incubated in absence of substrate exhibited an improved thermostability compared to its soluble counterpart, the half-life time at 80 or 90 °C being not detectable for 5 h or equal to 2 h for Eu–Cu and to 1.0 h or 0.3 h for the free enzyme, respectively. However, in bioconversions in three repeated cycles at 80 °C with 18 g L-1 of initial D-galactose, the immobilized biocatalyst activity declined rapidly. At 60 °C, not only Eu–Cu derivatives used in three repeated bioconversions with initial D-galactose concentration of 18 g L-1 were stable for at least 242 h, they also yielded a D-galactose conversion and an average productivity of 29.1% and 0.06 g L-1 h-1, respectively, exhibiting better performances compared to the results at 80 °C. [ABSTRACT FROM AUTHOR]

L-arabinose isomerase (L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The K m and k cat values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s -1 , respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose, as well as that of D-galactose to D-tagatose. A thermophilic AI derived from Lactobacillus fermentum CGMCC2921 (LFAI) was overexpressed in Escherichia coli BL21 (DE3). This enzyme was purified to over 95% purity by nickel affinity, Mono-Q ion-exchange and size-exclusion chromatography. The LFAI protein was crystallized from either 0.1 M bis-tris pH 6.5, 23% PEG 3350, 0.3 M NaCl (form 1 crystals) or 0.1 M bis-tris pH 6.0, 25% PEG monomethyl ether 5000 (form 2 crystals). Diffraction data from form 1 LFAI crystals were collected to 2.80 Å resolution using synchrotron radiation. The form 1 crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.11, b = 184.57, c = 186.26 Å, α = β = γ = 90°. The asymmetric unit contained six LFAI subunits, corresponding to a calculated Matthews coefficient of 2.29 Å3 Da−1 and a solvent content of 46.22%. [ABSTRACT FROM AUTHOR]

L-arabinose isomerase catalyzes the isomerization reaction from D-galactose to D-tagatose, and has attracted more attention due to its importance in animal nutrition and food applied research. Animal rumen is a special anaerobic environment, and ruminal microorganisms contain large amounts of novel genes that code for polysaccharide-degrading enzymes. This stud)' primarily analyzed the genetic diversity of L-arabinose isomerase in sheep rumen bacteria. Using the metagenome of rumen content extracted 4 h after feeding as template, 61 L-arabinose isomerase gene fragments with 68%~98% identities and known sequences were obtained by PCR amplification with specific degenerate primers and restriction enzyme digest. Of them, 38 distinct sequences (<95% identity) were subjected to clustering analysis. The results showed that the L-arabinose isomerase genes from Prevotella ruminicola were found to be predominant (36/38) in sheep rumen, and those from Catonella morbid and Marinilabilia salmonicolor (2/38) were minor. This study has not only enriched the genetic resources of L-arabinose isomerase, but also determined the importance of Prevotella in sheep rumen. [ABSTRACT FROM AUTHOR]

The double-site variant (C450S-N475K) l-arabinose isomerase ( l-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of d-galactose to d-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to d-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for d-galactose isomerization. The k/ K of the triple-site variant enzyme for d-galactose was 2.1-fold higher than for l-arabinose, whereas the k/ K of the double-site variant enzyme for l-arabinose was 43.9-fold higher than for d-galactose. These results suggest that the triple-site variant enzyme is a d-galactose isomerase. The conversion rate of d-galactose to d-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of l-arabinose to l-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for d-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for d-galactose and l-arabinose, and the triple-site variant enzyme has the potential to produce d-tagatose on an industrial scale. [ABSTRACT FROM AUTHOR]

L-Arabinose isomerase (AI), a key enzyme in the microbial pentose phosphate pathway, has been regarded as an important biological catalyst in rare sugar production. This enzyme could isomerize L-arabinose into L-ribulose, as well as D-galactose into D-tagatose. Both the two monosaccharides show excellent commercial values in food and pharmaceutical industries. With the identification of novel AI family members, some of them have exhibited remarkable potential in industrial applications. The biological production processes for D-tagatose and L-ribose (or L-ribulose) using AI have been developed and improved in recent years. Meanwhile, protein engineering techniques involving rational design has effectively enhanced the catalytic properties of various AIs. Moreover, the crystal structure of AI has been disclosed, which sheds light on the understanding of AI structure and catalytic mechanism at molecular levels. This article reports recent developments in (i) novel AI screening, (ii) AI-mediated rare sugar production processes, (iii) molecular modification of AI, and (iv) structural biology study of AI. Based on previous reports, an analysis of the future development has also been initiated. [ABSTRACT FROM AUTHOR]