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.

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 (kcat/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.

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.

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.

The biocatalysts are broadly explored in the biological transformation processes. The enzyme cascade catalysis involves various catalytic activities in a sequential process to produce the desired product including the formation of reaction intermediates. Enzyme immobilization is a method in which enzymes are confined within a support or matrix either physically or chemically to enhance their relative stability and catalytic activity in the enzyme cascade catalysis. In view of this, L-arabinose isomerase (L-AI) and L-ribose isomerase (L-RI) were immobilized on zeolite based metal framework as a micro-composite construct (DEMC@L-AI+L-RI) using linker, and metal ions. Such immobilization could be of great significance and provide several advantages like mesoporous surface for enzyme adsorption, desirable functionality in the production of products in enzyme cascade reaction, high storage stability and enhanced recyclability. The developed DEMC@L-AI+L-RI was characterized using SEM, FTIR, CLSM and TGA. The immobilization yield was 32% and loading of enzyme was 22% on the surface of micro-composite. The DEMC@L-AI+L-RI showed relatively stable catalytic activity at pH 5-6 and temperature 40 °C. The catalytic efficiency (kcat/Km) of both the enzymes was increased by 1.5-fold after immobilization. With the immobilized biocatalyst, bioconversion of L-arabinose to L-ribose was 22.6% and D-galactose to D-talose was 15.2%. The reusability of developed biocatalyst for more than six cycles was observed for more than 50% yield of the sugars. The conversion of biomass sugars from beetroot and onion waste residues was 20% and 14% to produce ribose and talose, respectively.

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.

d-Tagatose, a potential low calorific substitute for sucrose, can be produced by bioconversion of d-galactose catalysed by l-arabinose isomerase. l-Arabinose isomerase from Shewanella sp. ANA-3 is unique for its ability to catalyse bioconversion reactions under mesophilic conditions. However, d-galactose not being a natural substrate for l-arabinose isomerase is catalysed at a slower rate. We attempted to increase the biocatalytic efficiency of Shewanella sp. l-arabinose isomerase by rational design to enhance galactose isomerisation activity. In silico molecular docking, analysis has revealed that F279 is sterically hindering the binding of d-galactose at the C6 position. Substitution of bulky Phe residue with smaller hydrophilic residues such as Asn and Thr increased the galactose isomerase activity by 86 % and 12 % respectively. At mesophilic conditions, F279N mutant catalysed the bioconversion of d-galactose more efficiently than l-arabinose, indicating a shift in substrate preference.

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.

A dual-enzyme metal-organic hybrid crystal was constructed through self-assembling of manganese phosphate embedded with β-galactosidase and L-arabinose isomerase for facile synthesis of rare sugar D-tagatose. The synthesized crystal-like hierarchical system (MnHC@β-Gal+L-AI) was extensively characterized for structural features and catalytic reactions. The results indicated that upon immobilization onto the hybrid crystal, the activity of β-galactosidase and L-arabinose iomerase was enhanced by a factor of 1.6- and 1.5-fold, respectively. The developed MnHC@β-Gal+L-AI exhibited excellent efficiency with a net equilibrium level conversion of low-cost substrate whey lactose (100%) into D-glucose (∼50%), D-galactose (∼25%), and D-tagatose (∼25%). In addition, the fabricated hybrid crystals displayed cofactor regeneration ability. Therefore, the developed hybrid system was observed to be efficiently reused more than 5 times in a batch level conversion. Hence, the developed dual-enzyme-based hybrid crystal provides a platform for direct transformation of whey lactose into rare sugar D-tagatose.

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.