Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp.

For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (ee_p) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.     

Cloning, characterization and molecular docking of a highly thermostable β-1,4-glucosidase from Thermotoga petrophila

A genomic DNA fragment, encoding a thermotolerant β-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5?kDa protein (by SDS-PAGE) encoded by 1.341?kb gene. The estimated K (m) and V (max) values against p-nitrophenyl-β-D-glucopyranoside were 2.8?mM and 42.7?mmol?min(-1)?mg(-1), respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.     

Cloning,expression, and characterization of a thermostable β-xylosidase from thermoacidophilic Alicyclobacillus sp. A4

A β-xylosidase gene (xylA4) was identified in the genome sequence of thermoacidophilic Alicyclobacillus sp. A4. The deduced amino acid sequence was highly homologous with the β-xylosidases of family 52 of the glycoside hydrolases (GH). The full-length gene consisted of 2097 bp and encoded 698 amino acids without a signal peptide. The gene product was successfully expressed in Escherichia coli with an activity of 564.9 U/mL. Recombinant XylA4 was purified by Ni²⁺-NTA affinity chromatography with a molecular mass of 78.5 kDa. The enzyme showed optimal activity at pH 6.0 and 65 °C, and remained stable over the pH range of 5.0–9.0. The thermostability of XylA4 is noteworthy, retaining almost all of the activity after 1 h incubation at 65 °C. Using p-nitrophenyl-β-d-xylopyranoside (pNPX) as the substrate, XylA4 had the highest specific activity (261.1 U/mg) and catalytic efficiency (601.5/mM/s) known so far for GH52 xylosidases. The enzyme displayed high tolerance to xylose, with a K_i value of approximately 88.7 mM. It also had synergy with xylanase XynBE18 from Paenibacillus sp. E18 in xylan degradation, releasing more xylose (up to 1.43 folds) than XynBE18 alone. Therefore, this thermostable xylose-tolerant β-xylosidase may have a great application potential in many industrial fields.     

Substrate-dependent production and some properties of a thermostable, α-galactosidase from Rhodothermus marinus

-Galactosidase activity in Rhodothermus marinus is dependent on the composition of the growth media. A maximum of 46 U g^–1 cell dry weight was obtained using minimal medium with galactooligo- or polysaccharides as single carbon source. An enzyme hydrolysing both high and low molecular weight galacto-saccharides was partly purified from the cell fractions. The molecular weight was 200 kDa (native) and 50 kDa (monomer). It was optimally active at 85°C, with a half-life of 2 h at 75°C, and had a broad pH range (4–8).     

Cloning,expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13

A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co²⁺ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20?°C and pH of 8.3: k_cat of 4.8?×?10⁵?s^?1 and k_cat/K_m of 5.6?×?10⁷ M^?1?×?s^?1. This activity was potently inhibited by acetazolamide which showed a K_I of 78.9?nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO₃ biomineralization processes.     

Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry

Objectives

To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing.

Results

A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T ₅₀ value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k _cat value were 42,734 U mg^?1 and 189 s^?1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml^?1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively.

Conclusions

The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

     

Enzymatic production of Cilastatin intermediate via highly enantioselective hydrolysis of methyl (±)-2,2-dimethylcyclopropane carboxylate using newly isolated Rhodococcus sp. ECU1013

(S)-(+)-2,2-Dimethylcyclopropane carboxylic acid [(S)-(+)-DMCPA] is a key chiral intermediate for production of Cilastatin, an excellent renal dehydropeptidase-I inhibitor. In this study, a new method for preparation of (S)-(+)-DMCPA with microbial esterases was investigated. A microbial screening program obtained six esterase-producing isolates that could display relatively high activities and enantioselectivities using racemic ethyl 2,2-dimethylcyclopropane carboxylate (DMCPE) as screening substrate, aiming at forming optically pure (S)-(+)-DMCPA. Further selection was carried out with substrates having different alcohol moieties, including methyl, ethyl, and 2-chloroethyl esters. Finally, one of these strains, numbered ECU1013, with high enantioselectivity toward the hydrolytic resolution of methyl 2,2-dimethylcyclopropane carboxylate (DMCPM), afforded the (S)-product in 92 % ee, and was later identified as Rhodococcus sp. According to our research, there were several active esterases to DMCPM in cells of Rhodococcus sp. ECU1013; however, (S)-preferential esterase was selectively enriched based on the time-dependent profile of esterases biosynthesis, thereby the enantiomeric excess of biotransformation product (ee _p) was constantly increased, finally maintained at 95 % (S). To improve the yield, various organic solvents were employed for better dispersion of the hydrophobic substrate. As a result, (±)-DMCPM of up to 400 mM in the organic phase of isooctane was enantioselectively hydrolyzed into (S)-(+)-DMCPA, with an isolation yield of 38 % and a further increase of ee _p to 99 %.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

Effect of levulinic acid on cell growth and poly-β-hydroxyalkanoate production by Alcaligenes sp. SH-69

Summary Following growth of Alcaligenes sp. SH-69 on glucose as a sole carbon source for the production of poly--hydroxyalkanoates (PHAs), relatively low levels of levulinic acid (LA) were detected. Experiments were carried out in batch and continuous culture, and the effects of LA addition on growth and PHA synthesis were determined. Significant stimulatory effects were observed, greater than those for propionic acid addition. In N-limited two stage continuous culture, a maximal PHA content of 38.3 % (w/w) was achieved with a polyhydroxyvalerate (PHV) content of 23.5 % (molar basis) at D=0.078 l/h. This resulted from the controlled addition of LA at 0.5 g/L/h in the presence of excess glucose.     

Cloning and characterization of a new β-mannosidase from Streptomyces sp. S27

A new β-mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 °C. The specific activity, K_m, and k_cat values for p-nitrophenyl-β-d-mannopyranoside (p-NP-β-MP) were 35.3 U mg^-1, 0.23 mM, and 305 s^-1, respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP-β-MP (glycosyl donor) and methyl-α-d-mannopyranoside (p-NP-α-MP) (acceptor) at 50 °C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and β-mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions.     

Preparation of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid and its derivatives from racemates by enantioselective degradation with a newly isolated bacterial strain <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ECU0401

An enantioselective mandelate-degrading bacterium, Alcaligenes sp. ECU0401, was newly isolated from soil. By fed-batch culture, (R)-(-)-mandelic acid was successfully prepared in a 5-L fermenter with 32.8% isolated yield and >99.9% enantiomeric excesses (e.e.) from totally 3.04% (w/v) of racemic mandelic acid after 99 h of biotransformation. The optimal reaction pH and temperature were 6.5 and 30 degrees C, respectively. Using the resting cell as a biocatalyst for asymmetric degradation of racemic mandelic acid and chloro-substituted derivatives thereof, (R)-(-)-mandelic acid, (R)-(-)-o-chloromandelic acid, (S)-(+)-m-chloromandelic acid and (S)-(+)-p-chloromandelic acid were recovered with high analytic yields and excellent enantiomeric excesses (e.e. > 99.9%). (R)-(-)-Mandelic acid could also be obtained after 12 h of biotransformation with 41.5% isolated yield and >99.9% e.e.     

Bioprocess exploration for thermostable α-amylase production of a deep-sea thermophile Geobacillus sp. in high-temperature bioreactor

Geobacillus sp. 4j, a deep-sea high-salt thermophile, was found to produce thermostable α-amylase. In this work, culture medium and conditions were first optimized to enhance the production of thermostable α-amylase by statistical methodologies. The resulting extracellular production was increased by five times and reached 6.40?U/ml. Then, a high-temperature batch culture of the thermophile in a 15?l in-house-designed bioreactor was studied. The results showed that a relatively high dissolved oxygen (600?rpm and 15?l/min) and culture temperature of 60°C facilitated both cell growth and α-amylase production. Thus, an efficient fermentation process was established with initial medium of pH 6.0, culture temperature of 60°C, and dissolved oxygen above 20%. It gave an α-amylase production of 79?U/ml and productivity of 19804?U/l·hr, which were 10.8 and 208 times higher than those in shake flask, respectively. This work is useful for deep-sea high-salt thermophile culture, where efforts are lacking presently.     

Purification and characterization of a thermostable λ-carrageenase from a hot spring bacterium,Bacillus sp.

Purpose of work The purpose of this study is to report a thermostable λ-carrageenase that can degrade λ-carrageenan yielding neo-λ-carrabiose at 75 °C. A thermophilic strain Lc50-1 producing λ-carrageenase was isolated from a hot spring in Indonesia and identified as a Bacillus sp. The λ-carrageenase, Cga-L50, with an apparent molecular weight of 37 kDa and a specific activity of 105 U/mg was purified from the culture supernatant. The optimum pH and temperature of Cga-L50 were 8.0 and 75 °C, respectively. The enzyme was stable from pH 6–9 and retained ~50 % activity after holding at 85 °C for 10 min. Significant activation of Cga-L50 was observed with K⁺, Ca²⁺, Co²⁺, and Na⁺; whereas, the enzyme activity was inhibited by Sr²⁺, Mn²⁺, Fe²⁺, Cu²⁺,Cd²⁺, Mg²⁺, and EDTA. Cga-L50 is an endo-type λ-carrageenase that hydrolyzes β-1,4-linkages of λ-carrageenan, yielding neo-λ-carrabiose as the main product. This study is the first to present evidence of thermostable λ-carrageenase from hot spring bacteria.     

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V _max and K _m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 μmol mg^?1 min^?1 and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K _i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml ^? resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.     

Purification and some properties of cellulose 1,4-β-cellobiosidase from a strain of Penicillium sp.

A cellulase was purified from the culture supernatant of a strain of Penicillium sp. The purified enzyme was homogenous on polyacrylamide disc gel electrophoresis. It was a glycoprotein with a molecular weight of 52,000 estimated by gel filtration. The optimum pH was about 4.0 and the optimum temperature was 60°C. The enzyme was stable in the pH range of 3.0–10.0 at 6°C for 48 h and on heating at 60°C for 10 min. The activity of the enzyme toward Avicel was about 3 times higher than toward carboxymethyl cellulose. The enzyme showed a low activity for cotton, newspaper, filter paper and cellulose powder. The main product from Avicel was cellobiose, with a trace of glucose.     

Isolation of a fosfomycin-hypersensitive mutant and production of α-d-glucose-l-phosphate from Bacillus sp. BA-3796 screened by its use

The development of a very sensitive and highly specific screening method for detection of new cell wall inhibitors of the fosfomycin type is described. A fosfomycin-hypersensitive mutant, f-360, was isolated from Staphylococcus aureus Newman by selection with fosfomycin, an antibiotic that inhibits synthesis of the bacterial cell wall. The mutant f-360 was 50-fold more sensitive than the parent strain to fosfomycin. The mutant was constitutive for the hexose phosphate transport system. Using the organism in screening, BA-3796, which had an antibacterial activity against mutant f-360 was found to be produced by a bacterium designated Bacillus sp. BA-3796. Starch and beef extract were the most suitable carbon and nitrogen sources for BA-3796 production and the amount of BA-3796 reached 3 g/l at a maximum level. The purified BA-3796 was identified as α-d-glucose-l-phosphate by its various physiochemical properties. α-d-Glucose-1-phosphate showes an antibacterial activity against Staphylococci in the presence of a slight amount pf α-d-glucose-6-phosphate.     

Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum

The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties, such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4 M glucose/1.0 M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-d-glucopyranoside as substrate the values were Vmax (7614 U/mg), and K_M (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and K_M 0.877 mM (glucose), and 1.410 mM (xylose).     

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure–function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.     

Cloning,expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1

Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β-d-galactopyranoside but not p-nitrophenyl-α-d-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K _m and V _max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and ¹³C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.