Alginate immobilization of recombinant Escherichia coli whole cells harboring l-arabinose isomerase for l-ribulose production

Recombinant Escherichia coli whole cells harboring Bacillus licheniformis l-arabinose isomerase (BLAI) were immobilized with alginate. The operational conditions for immobilization were optimized with response surface methodology. Optimal alginate concentration, Ca²⁺ concentration, and cell mass loading were 1.8% (w/v), 0.1 M, and 44.5 g L⁻¹, respectively. The interactions between Ca²⁺ concentration, alginate concentration, and initial cell mass were significant. After immobilization of BLAI, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The half-life of immobilized whole cells was 150 days, which was 50-fold longer than that of free cells. In seven repeated batches for l-ribulose production, the productivity was as high as 56.7 g L⁻¹ h⁻¹ at 400 g L⁻¹ substrate concentration. The immobilized cells retained 89% of the initial yield after 33 days of reaction. Immobilization of whole cells harboring BLAI, therefore, makes a suitable biocatalyst for the production of l-ribulose, particularly because of its high stability and low cost.     

Stabilization of d-amino acid oxidase from Rhodosporidium toruloides by immobilization onto magnetic nanoparticles

d-Amino acid oxidase from Rhodosporidium toruloides was immobilized onto glutaraldehyde-activated magnetic nanoparticles. Approximately four enzyme molecules were attached to one magnetic nanoparticle when the weight ratio of the enzyme to the support was 0.12. After immobilization, the T _m was increased from 45°C of the free form to 55°C. In the presence of 20 mM H₂O₂, the immobilized form retained 93% of its activity after 5 h while the free form was completely inactivated after 3.5 h.     

Stabilization of native and double <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidases from <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> and <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis> by immobilization on streptavidin-coated magnetic beads

Double d-amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k_cat/K_M) of immobilized DAOs toward d-alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward d-alanine was decreased by 56%. After immobilization, the T_m value for RtDAO was shifted 15°C higher to 60°C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5–8°C to 56, 60 and 60°C, respectively. In the presence of 10 mM H₂O₂, immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin–streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.     

Immobilization of Bacillus licheniformis L-Arabinose Isomerase for Semi-Continuous L-Ribulose Production

Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 °C (free enzyme) to a range between 55 and 65 °C. The half life improved from 2 at 50 °C to 212 h at 55 °C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m², however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m². The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K_m) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V_max) remained almost constant.     

Covalent immobilization of β-1,4-glucosidase from <Emphasis Type="Italic">Agaricus arvensis</Emphasis> onto functionalized silicon oxide nanoparticles

An efficient β-1,4-glucosidase (BGL) secreting strain, Agaricus arvensis, was isolated and identified. The relative molecular weight of the purified A. arvensis BGL was 98 kDa, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. Using a crude enzyme preparation, A. arvensis BGL was covalently immobilized onto functionalized silicon oxide nanoparticles with an immobilization efficiency of 158%. The apparent V _max (k _cat) values of free and immobilized BGL under standard assay conditions were 3,028 U mg protein⁻¹ (4,945 s⁻¹) and 3,347 U mg protein⁻¹ (5,466 s⁻¹), respectively. The immobilized BGL showed a higher optimum temperature and improved thermostability as compared to the free enzyme. The half-life at 65 °C showed a 288-fold improvement over the free BGL. After 25 cycles, the immobilized enzyme still retained 95% of the original activity, thus demonstrating its prospects for commercial applications. High specific activity, high immobilization efficiency, improved stability, and reusability of A. arvensis BGL make this enzyme of potential interest in a number of industrial applications.     

Expression,purification and immobilization of the intracellular invertase INVA,from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on crystalline cellulose and Nylon-6

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD _Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K _m and V _max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V _max increased up to 5.7-fold, following immobilization, whereas K _m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.     

Cysteine enhances activity and stability of immobilized papain

Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. After immobilization, the optimum temperature of papain activity increased by 20°C (from 60 to 80°C) and its optimum pH activity shifted from 6.5 to 8.0. Catalytic efficiency (k_cat/K_m) and specific activity of the immobilized enzyme do not significantly change after immobilization. The temperature profile of this form of immobilized papain showed a broad range of activity compared with both free and immobilized form of papain in the absence of cysteine. This significant behavior in terms of activation energy is also discussed.     

Increased <Emphasis Type="SmallCaps">d</Emphasis>-allose production by the R132E mutant of ribose-5-phosphate isomerase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k _cat/K _m) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

One-step purification and immobilization of thermophilic polyphosphate glucokinase from <Emphasis Type="BoldItalic">Thermobifida fusca</Emphasis> YX: glucose-6-phosphate generation without ATP

The discovery of stable and active polyphosphate glucokinase (PPGK, EC 2.7.1.63) would be vital to cascade enzyme biocatalysis that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was overexpressed in Escherichia coli. Mg²⁺ was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg²⁺ at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k _cat and K _m values of CBM-PPGK on glucose were 96.9 and 39.7 s⁻¹ as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose, immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t _1/2 = 120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported. Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively, suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate.     

Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.     

α-<Emphasis Type="SmallCaps">l</Emphasis>-Rhamnosidase of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> immobilized on ferromagnetic supports

α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K _m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).     

A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

A novel method was developed for the immobilization of glucoamylase from Aspergillus niger. The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K_m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V_max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.     

Covalent immobilization of recombinant <Emphasis Type="Italic">Rhizobium etli</Emphasis> CFN42 xylitol dehydrogenase onto modified silica nanoparticles

Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to give different functional groups, the silica nanoparticle derivatized with epoxy groups showed the highest immobilization efficiency (92%). The thermostability of ReXDH was improved more than tenfold by immobilization on epoxy-silica nanoparticles; the t _1/2 of the ReXDH was enhanced from 120 min to 1,410 min at 40 °C and from 30 min to 450 min at 50 °C. The K _m of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into l-xylulose. A ∼ 71% conversion and a productivity of 10.7 g h^-1 l^-1 were achieved when the immobilized ReXDH was employed to catalyze the biotransformation of xylitol to l-xylulose, a sugar that has been used in medicine and in the diagnosis of hepatitis. These results suggest that immobilization of ReXDH onto epoxy-silica nanoparticles has potential industrial application in rare sugar production.     

Performance of Aspergillus niger B 03 β-xylosidase immobilized on polyamide membrane support

The dynamics of β-xylosidase biosynthesis from Aspergillus niger B 03 was investigated in laboratory bioreactor. Maximum xylosidase activity 5.5 U/ml was achieved after 80 h fermentation at medium pH 4.0. The isolated β-xylosidase was immobilized on polyamide membrane support and the basic characteristics of the immobilized enzyme were determined. Maximum immobilization and activity yield obtained was 30.0 and 6.8%, respectively. A shift in temperature optimum and pH optimum was observed for immobilized β-xylosidase compared to the free enzyme. Immobilized enzyme exhibited maximum activity at 45 °C and pH 4.5 while its free counterpart at 70 °C and pH 3.5, respectively. Thermal stability at 40 and 50 °C and storage stability of immobilized β-xylosidase were investigated at pH 5.0. Kinetic parameters K_m, V_max and K_i were determined for both enzyme forms. Free and immobilized β-xylosidase were tested for xylose production from birchwood xylan. The substrate was preliminarily depolymerized with xylanase to xylooligosaccharides and the amount of xylose obtained after their hydrolysis with free and immobilized β-xylosidase was determined by HPLC analysis. Continuous enzyme hydrolysis of birchwood xylan was performed with xylanase and free or immobilized β-xylosidase. The maximum extent of hydrolysis was 25 and 30% with free and immobilized enzyme, respectively. Immobilized preparation was also examined for reusability in 20 consecutive cycles at 40 °C.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.