The addition of Co2+ enhances the catalytic efficiency and thermostability of recombinant glucose isomerase from Thermobifida fusca

Glucose isomerase is an important industrial enzyme that catalyzes the reversible isomerization of glucose to fructose. In this study, the effect of cobalt ions (Co²⁺) on the catalytic efficiency and thermostability of recombinant glucose isomerase from Thermobifida fusca was analyzed. The activity of glucose isomerase from engineered Escherichia coli supplemented with 1 mM Co²⁺ (C-GI) reached 41 U/ml, 2.1-fold higher than enzyme prepared from E. coli without additive (GI). The purified C-GI also exhibited an increased specific activity (23.8 U/mg compared to 12.1 U/mg for GI) and a greater thermostability (half-life of 17 h at 75 °C, 11.3-fold higher than GI (1.5 h)). The optimal temperature for C-GI shifted from 80 °C to 85 °C and demonstrated higher activity over pH 7.0–9.0. The k_cat/K_m value of C-GI (89.3 M^?1 s^?1) for the isomerization of glucose to fructose was nearly 1.75-fold higher than that of GI. In addition, the engineered cells were immobilized with the method of flocculation-cross linking. The immobilized cells supplemented with 1 mM Co²⁺ (C-IGI) had a better operational performance than cells without additives (IGI); at the end of 6 cycles, the conversion rate of C-IGI was still 43.1%, meeting the conversion rate requirement.     

Improvement and characterization of a hyperthermophilic glucose isomerase from <Emphasis Type="Italic">Thermoanaerobacter</Emphasis><Emphasis Type="Italic">ethanolicus</Emphasis> and its application in production of high fructose corn syrup

High fructose corn syrup (HFCS) is an alternative of liquid sweetener to sucrose that is isomerized by commercial glucose isomerase (GI). One-step production of 55 % HFCS by thermostable GI has been drawn more and more attentions. In this study, a new hyperthermophilic GI from Thermoanaerobacter ethanolicus CCSD1 (TEGI) was identified by genome mining, and then a 1317 bp fragment encoding the TEGI was synthesized and expressed in Escherichia coli BL21(DE3). To improve the activity of TEGI, two amino acid residues, Trp139 and Val186, around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected for site-directed mutagenesis. The specific activity of mutant TEGI-W139F/V186T was 2.3-fold and the value of k _cat/K _m was 1.86-fold as compared to the wild type TEGI, respectively. Thermostability of mutant TEGI-W139F/V186T at 90 °C for 24 h showed 1.21-fold extension than that of wild type TEGI. During the isomerization of glucose to fructose, the yield of fructose could maintain above 55.4 % by mutant TEGI-W139F/V186T as compared to 53.8 % by wild type TEGI at 90 °C. This study paved foundation for the production of 55 % HFCS using the thermostable TEGI.     

葡萄糖异构酶及其在高果糖浆生产中的应用

葡萄糖异构酶(Glucose isomerase,GI)能催化D-葡萄糖的异构化反应,生成D-果糖,是目前工业上制备高果糖浆(HFCS)的关键酶之一。本文对GI的来源、分类、高级结构特征和催化机制进行了介绍,并从GI催化功能的改善、基因工程菌的构建和固定化三个方面对GI在HFCS生产中应用的关键技术和策略进行分析。     

Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.     

Purification and characterization of a highly selective sucrose isomerase from Erwinia rhapontici NX-5

A highly selective sucrose isomerase (SIase) was purified to homogeneity from the cell-free extract of Erwinia rhapontici NX-5 with a recovery of 27.7% and a fold purification of 213.6. The purified SIase showed a high specific activity of 427.1 U mg⁻¹ with molecular weight of 65.6 kDa. The K _m for sucrose was 222 mM while V _max was 546 U mg⁻¹. The optimum pH and temperature for SIase activity were 6.0 and 30 °C, respectively. The purified SIase was stable in the temperature range of 10–40 °C and retained 65% of the enzyme activity after 2 weeks’ storage at 30 °C. The SIase activity was enhanced by Mg²⁺ and Mn²⁺, inhibited by Ca²⁺, Cu²⁺, Zn²⁺, and Co²⁺, completely inhibited by Hg²⁺ and Ag²⁺. The purified SIase was strongly inhibited by SDS, while partially inhibited by dimethylformamide, tetrahydrofuran, and PMSF. Additionally, glucose and fructose acted as competitive inhibitors for purified SIase.     

Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g⁻¹ dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g⁻¹ dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m⁻² s⁻¹ had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g⁻¹ dry weight in darkness to 20 mg g⁻¹ dry weight at a light intensity of 80 μmol photons m⁻² s⁻¹. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day⁻¹, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g⁻¹ dry weight was found at 65 μmol photons m⁻² s⁻¹. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.     

Production,purification and characterization of a novel GH 12 family endoglucanase from Aspergillus terreus and its application in enzymatic degradation of delignified rice straw

Endoglucanase production was carried out using in-house isolate Aspergillus terreus on rice straw under solid state fermentation. An increase of 1.25-fold endoglucanase production was obtained under optimized conditions using response surface methodology. The enzyme was purified to homogeneity by gel filtration chromatography. Its molecular weight was determined as 28.18 kDa by gel filtration and 29.13 kDa on SDS-PAGE. The enzyme displayed maximum activity at 50 °C and pH 4.8. It was stable for 240 min at 50 °C and 120 min at 60 °C but rapidly inactivated at 70 °C. The purified enzyme was specific towards carboxymethyl-cellulose but showed no activity for cellobiose or xylan. Maximum velocity (V_max) and K_M were 16.15 μmol min⁻¹ mg⁻¹ and 12.01 mg ml⁻¹, respectively. AgNO₃, KCl, NaCl, and MnSO₄ were found to inhibit enzyme activity while CaCl₂ and ZnSO₄ activated the enzyme. Internal peptide mass fingerprinting analysis identified that the protein belongs to GH12 superfamily endoglucanases. External supplementation of the purified enzyme to the crude cellulase showed 38.7% increase in saccharification efficiency of the delignified rice straw compared to the crude cellulase alone. The results demonstrated that the addition of GH 12 family purified endoglucanase to the crude cellulase can efficiently convert lignocellulosic biomass to fermentable sugars.     

Kinetics of glucose isomerization to fructose by immobilized glucose isomerase in the presence of substrate protection

The activity of immobilized glucose isomerase of Streptomyces murinus has been tested batchwise under different conditions in order to gather the related kinetic parameters necessary to optimize an immobilized enzyme column for the continuous production of high fructose corn syrup (HFCS). To this purpose, the Briggs-Haldane model incorporating an apparent first-order inactivation constant has been used with success. A comparison of the equilibrium constants and of the maximum theoretical conversion yields calculated at different temperatures with those estimated for the native enzyme demonstrates that the immobilization favours the transformation of glucose to fructose only at T?>?70?°C, as a possible consequence of a combined effect of catalysis and equilibrium thermodynamics enhancement. Enzyme inactivation has also been tested at different temperatures and sugar concentrations to evaluate the related kinetic parameters under different conditions of substrate protection.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation

The pectinolytic enzyme obtained from Penicillium viridicatum RFC by solid-state fermentation was purified to homogeneity by pretreatment with kaolin (40 mg mL⁻¹) and ultrafiltration, followed by chromatography on a Sephadex G50 column. The apparent molecular weight of the enzyme was 24 kDa. Maximal activity occurred at pH 6.0 and at 60 °C. The enzyme proved to be an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of highly esterified pectin. The presence of 10 mM Ba²⁺ increased the enzyme activity by 96% and its thermal stability by 30%, besides increasing its stability at acid pH. The apparent K_m with apple pectin as substrate was 1.82 mg mL⁻¹ and the V_max was 81 μmol min⁻¹ mg⁻¹.     

Treatment of dye-rich wastewater by an immobilized thermophilic cyanobacterial strain: Phormidium sp.

The removal of Remazol Blue and Reactive Black B by the immobilized thermophilic cyanobacterial strain Phormidium sp. was investigated under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye removal. In the experiments, performed at pH 8.5, with different initial dye concentrations between 9.1 mg l⁻¹ and 82.1 mg l⁻¹ and at 45 °C, calcium alginate immobilized Phormidium sp. showed high dye decolorization, with maximum uptake yields ranging from 50% to 88% at all dye concentrations tested. When the effects of high dye concentrations on dye removal were investigated, the highest uptake yield in the beads was 50.3% for 82.1 mg l⁻¹ Remazol Blue and 60.0% for 79.5 mg l⁻¹ Reactive Black B. The highest color removal was detected at 45 °C and 50 °C incubation temperatures for all dye concentrations. As the temperature decreased, the removal yield of immobilized Phormidium sp. also decreased. At about 75 mg l⁻¹ initial dye concentrations, the highest specific dye uptake measured was 41.29–41.17 mg g⁻¹ for Remazol Blue and 47.69–43.82 mg g⁻¹ for Reactive Black B at 45 °C and 50 °C incubation temperatures, respectively, after 8 days incubation.     

Generalized linearization of kinetics of glucose isomerization to fructose by immobilized glucose isomerase

The kinetic parameters of both glucose isomerization to fructose and immobilized glucose isomerase (GI) inactivation calculated under different conditions are compared and discussed. Utilizing these figures, the possibility of generalizing a linear model, previously proposed for the kinetics of glucose isomerization by immobilized glucose isomerase, is investigated, so as to apply them to whole ranges of temperature and concentrations of actual interest in industrial processes. The proposed model is a satisfactory approximation of the more involved Briggs-Haldane approach and substantially simplifies the problem of optimizing an industrial fixed-bed column for high-fructose corn syrup (HFCS) production.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures

Four immobilized forms of glucose oxidase (GOD) were used for biotransformation removal of glucose from its mixture with dextran oligosaccharides. GOD was biospecifically bound to Concanavalin A-bead cellulose (GOD-ConA-TBC) and covalently to triazine-bead cellulose (GOD-TBC). Eupergit C and Eupergit CM were used for preparation of other two forms of immobilized GOD: GOD-EupC and GOD-EupCM. GOD-ConA-TBC and GOD-EupC exhibited the best operational and storage stabilities. pH and temperature optima of these two immobilized enzyme forms were broadened and shifted to higher values (pH 7 and 35 °C) in comparison with those of free GOD. The decrease of V_max values after immobilization was observed, from 256.8 ± 7.0 μmol min⁻¹ mg_GOD⁻¹ for free enzyme to 63.8 ± 4.2 μmol min⁻¹ mg_GOD⁻¹ for GOD-ConA-TBC and 45 ± 2.7 μmol min⁻¹ mg_GOD⁻¹ for GOD-EupC, respectively. Depending on the immobilization mode, the immobilized GODs were able to decrease the glucose content in solution to 3.8–15.6% of its initial amount The best glucose conversion, was achieved by an action of GOD-EupCM on a mixture of 100 g dextran with 9 g of glucose (i.e. 98.7% removal of glucose).     

Evidence for extracellular enzymic activity of the isolated perfused rat heart

1. The dissimilation of a number of externally added hexose phosphates and 5′-nucleotides by the perfused rat heart is described, and non-specific esterase and 5′-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of ¹⁴CO₂ from [U-¹⁴C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-¹⁴C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-¹⁴C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.     

Comparison of cold hardiness and sugar content between diapausing and nondiapausing pupae of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract. To understand overwintering of the cotton boll worm Helicoverpa armigera, cold hardiness and sugar content are compared between diapausing and nondiapausing pupae. Diapausing and nondiapausing pupae reared at 20 °C under short and long photoperiods are acclimatized with a reduction of 5 °C per 5 days to 0 °C. When the acclimation temperature reaches 0 °C, the survival of diapausing pupae is assessed. The survival gradually decreases as the period of treatment progresses and approximately half survive for 112 days. However, nondiapausing pupae survive only 14 days after exposure to 0 °C. The surpercooling points of nondiapausing, diapausing and acclimatized pupae are approximately −17 °C. The major sugars contained in pupae are trehalose and glucose. Even though trehalose contents in diapausing pupae (initial level: 0.6 mg 100 mg⁻¹ fresh weight) increase significantly during cold acclimation and continue increasing until 58 days after exposure to 0 °C (maximum level: 1.8 mg 100 mg⁻¹), glucose is maintained at low levels (0.02 mg 100 mg⁻¹) for 56 days at 0 °C. However, glucose contents increase (maximum level: 0.8 mg 100 mg⁻¹) with decreasing contents of trehalose 84 days after exposure to 0 °C. Glycogen content gradually decreases during cold acclimation. When nondiapausing pupae are acclimatized with a reduction of 5 °C per 5 days to 5 °C from the beginning of pupation until the eyespots move, trehalose content increases (maximum level: 1.0 mg 100 mg⁻¹). Glucose contents in nondiapausing pupae increase before eclosion (0.09 mg 100 mg⁻¹). From these results, diapausing pupae of H. armigera can overwinter in regions where average winter temperatures are higher than 0 °C, but nondiapausing pupae cannot.     

Molecular and industrial aspects of glucose isomerase.

Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the presence of two signature sequences, VXW(GP)GREG(YSTAE)E and (LIVM)EPKPX(EQ)P. The use of an inexpensive inducer in the fermentation medium devoid of Co2+ and redesigning of a tailor-made GI with increased thermostability, higher affinity for glucose, and lower pH optimum will contribute significantly to the development of an economically feasible commercial process for enzymatic isomerization of glucose to fructose. Manipulation of the GI gene by site-directed mutagenesis holds promise that a GI suitable for biotechnological applications will be produced in the foreseeable future.     

Influence on dithiolopyrrolone antibiotic production by organic acids in Saccharothrix algeriensis NRRL B-24137

The influence of organic acids on growth and dithiolopyrrolone antibiotic production by Saccharothrix algeriensis NRRL B-24137 was studied. The production of dithiolopyrrolones depends upon the nature and concentration of the organic acids in the culture medium. Study of the nature of organic acids showed that the most effective organic acids for thiolutin specific production were maleic, 4-hydroxybenzoic, benzentetracarboxylic, pantothenic, pivalic and pyruvic acids (which yielded almost five-fold over the starting medium) and pimelic acid (more than three-fold). 4-Bromobenzoic acid showed the best production of senecioyl-pyrrothine (59 mg g⁻¹ DCW). Tiglic acid showed the best production of tigloyl-pyrrothine (22 mg g⁻¹ DCW). The highest yield of isobutyryl-pyrrothine (7.6 mg g⁻¹ DCW) was observed in the presence of crotonic acid. Sorbic acid yielded the best production of butanoyl-pyrrothine (26 mg g⁻¹ DCW). Methacrylic, butyric, pyruvic and 4-bromobenzoic acids also exhibited the best production of butanoyl-pyrrothine (27–11-fold).Study of organic acid concentration showed that among the selected organic acids, pimelic acid yielded the highest specific production of thiolutin (91 mg g⁻¹ DCW) at 7.5 mM; and senecioyl-pyrrothine (11 mg g⁻¹ DCW), tigloyl-pyrrothine (9 mg g⁻¹ DCW) and butanoyl-pyrrothine (3.5 mg g⁻¹ DCW) at 5 mM. Pyruvic acid at 1.25 mM enhanced the production of senecioyl-pyrrothine (4.3 mg g⁻¹ DCW). The maximum production of tigloyl-pyrrothine (18.6 mg g⁻¹ DCW) was observed in the presence of tiglic acid at 2.5 mM. Maximum production of isobutyryl-pyrrothine was observed in the presence of 7.5 mM tiglic acid. In addition, methacrylic acid (at 5 mM) and butyric acid (at 2.5 mM) enhanced the production of butanoyl-pyrrothine (26 and 20 times, respectively).The above results can be employed in the optimisation of the culture medium for the production of dithiolopyrrolone in higher quantities.     

Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris

A β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris under the control of the AOX1 promoter. The coding region of 3036 bp encoded a protein of 1011 amino acids with a deduced molecular mass of 108.7 kDa. The PaGalA without the signal peptide was cloned into a vector pPIC9K and was expressed successfully in P. pastoris as active extracellular β-galactosidase. The recombinant β-galactosidase (PaGalA) was secreted into the medium at an extremely high levels of 22 mg ml⁻¹ having an activity of 9500 U ml⁻¹ from high density fermentation culture, which is by far the highest yield obtained for a β-galactosidase. The purified enzyme with a high specific activity of 820 U mg⁻¹ had a molecular mass of 120 kDa on SDS-PAGE. PaGalA was optimally active at pH 4.5 and a temperature of 60 °C. The recombinant β-galactosidase was able to hydrolyze lactose efficiently at pH 5.0 and 50 °C. It also possessed transglycosylation activities at high concentrations of lactose. PaGalA exhibited better lactose hydrolysis efficiency in whey than two other widely used commercial lactases. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.     

Characterization of recombinant L-ribose isomerase acquired from Cryobacterium sp. N21 with potential application in L-ribulose production

l-Ribose isomerase (lRI) is an enzyme that can catalyze the reversible isomerization between l-ribose and l-ribulose. It can also perform the conversion between many aldoses into their corresponding ketoses. l-RI was produced from Cryobacterium sp. N21 (CrL-RIse), and l-ribose was utilized as a substrate. The recombinant l-RI gene was cloned and overexpressed from Cryobacterium sp. N21. The purification of CrL-RIse was performed by metal-affinity chromatography. The enzyme displayed a corresponding band with an approximate size of 35 kDa on the SDS-PAGE analysis. The protein for this gene contains 266 amino acids with an expected molecular weight (M_w) of 29.6 kDa. The measured M_w of CrL-RIse calculated by HPLC was 125 kDa. CrL-RIse was extremely active in glycine buffer at 35 °C, pH 9.0, showing a specific activity of 54.96 U mg⁻¹. CrL-RIse displayed no major increase in activity with metal ions, excluding Mn²⁺. The estimated Km, K_cat, K_cat/Km and V_max values of CrL-RIse were 37.8 mM, 10,416 min⁻¹, 275.43 min⁻¹ mM⁻¹, and 250 U mg⁻¹, respectively. The rate of l-ribulose production was 31 % (6.24, 12.11, and 20.89 g L⁻¹) at equilibrium by utilizing 20, 40, and 70 g L⁻¹ of the substrate, respectively. The results indicated that CrL-RIse has the capability to manufacture l-ribulose from l-ribose.