Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation

A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest immobilization yield and stability being selected for the optimization of immobilization conditions and application in GOS synthesis. A central composite rotatable design was conducted to optimize contacted protein and immobilization time, using maximum catalytic potential as the objective function. Optimal conditions of immobilization were 28.9 mg/g and 36.4 h of contact, resulting in a biocatalyst with 595 IU/g and a half-life 89-fold higher than soluble enzyme. Immobilization process did not alter the synthetic capacity of β-galactosidase, obtaining the same GOS yield and product profile than the free enzyme. GOS yield and productivity remained unchanged along 10 repeated batches, with values of 39% (w/w) and 5.7 g GOS/g of biocatalyst·batch. Total product obtained after 10 batches of reaction was 56.5 g GOS/g of biocatalyst (1956 g GOS/g protein). Cumulative productivity in terms of mass of contacted protein was higher for the immobilized enzyme than for its soluble counterpart from the second batch of synthesis onwards.     

Acyl transfer strategy for the biocatalytical characterisation of Candida rugosa lipases in organic solvents

The yield obtained in a Candida rugosa lipase-catalysed heptyl acetate synthesis via transesterification of 1-heptanol with vinyl acetate depends linearly on the concentration of lipases of each crude lyophilised powder. Thus, it is possible to characterise the amount of lipases present in the biocatalyst by calculating the parameter called Catalytic Performance (CP), defined as (% final yield) × (mg crude biocatalyst)⁻¹. On the other hand, the relative yield obtained in the C. rugosa lipase-catalysed transesterifications of different alcohols (1-heptanol, geraniol, nerol and cyclohexanol) with vinyl acetate as acyl donor depends on the proportion of isoenzymes of each crude biocatalyst. Therefore, it is possible to qualitatively evaluate the proportion of isoenzymes in those crude preparations and to predict the biocatalytical behaviour of each isoenzyme according to the alcohol employed. The methodology described is successfully used in two non-conventional asymmetric syntheses in organic media.     

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8 ± 8.60 IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3 ± 1.70 mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50 °C, while for the crude PPL extract were at pH 8.0 and 45 °C. Immobilized PPL exhibited full hydrolytic activity after 2 h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2 h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48 h of reaction for MPKO and 35% after 96 h of reaction for MPPO.     

Biocatalytic synthesis of ethyl (R)-2-hydroxy-4-phenylbutyrate with Candida krusei SW2026: A practical process for high enantiopurity and product titer

Ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE), a key intermediate in the production of angiotensin-converting enzyme (ACE) inhibitors, was prepared by the microbial reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE). Among 63 microorganisms tested, Candida krusei SW2026, for the first time, was proven to be a highly effective biocatalyst in this reduction process, leading to the (R)-enantiomer in 99.7% ee and 95.1% yield at 2.5 g/L of OPBE (under optimal conditions of 30 °C, pH 6.6, and in the presence of 5% glucose as co-substrate). In order to achieve higher product concentration with desired enantiopurity and yield for application in large-scale production, strategies such as substrate fed-batch and aqueous/organic biphasic system were successfully conducted in the biotransformation reaction. At 20 g/L of OPBE, the enantiomeric excess (ee), yield, and product concentration were enhanced to 97.4%, 82.0%, and 16.6 g/L, respectively, in water/dibutyl phthalate biphasic system, compared with 87.5%, 45.8%, and 9.2 g/L in aqueous medium. This study provides an attractive process of (R)-HPBE production for potential green chemistry applications.     

Development of a recombinant Escherichia coli-based biocatalyst to enable high styrene epoxidation activity with high product yield on energy source

A highly active recombinant whole-cell biocatalyst, Escherichia coli pETAB2/pG-KJE1, was developed for the efficient production of (S)-styrene oxide from styrene. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL–GroES and DnaK–DnaJ–GrpE). The styrene monooxygenases were produced to ca. 40% of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180 U/g CDW. The high StyAB activity in turn appeared to direct cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40%. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 6.4 mmol/g dry cells/h (106 U/g dry cells) and 67 mM/h (1110 U/L_aq), respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli

An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use.     

Lipase-catalyzed synthesis of capsiate analog using vanillyl alcohol and conjugated linoleic acid under vacuum system

This study focused on the application of vacuum system to synthesize capsiate analogs. The capsiate analogs containing conjugated linoleic acid (CLA) was successfully synthesized in solvent free system via lipase-catalyzed esterification. This esterification was carried out using vanillyl alcohol and CLA as substrates, and Lipozyme RM IM from Rhizomucor miehei as a biocatalyst. The best reaction condition was a molar ratio of 1:2 (vanillyl alcohol to CLA), a reaction temperature of 50 °C, and a lipase loading of 10% (w/w, based on total substrates). Application of vacuum increased the yield of capsiate analog as well as the reaction rate. When the vacuum levels were between 66.7 kPa and 1.3 kPa, an equilibrium yield of 100 mol% was achieved. The maximum yield was approached after only 3 h of reaction at the vacuum levels of higher than 13.3 kPa. The content of 9c,11t-CLA in capsiate analog synthesized was higher than that of 10t,12c-CLA.     

Bio-resolution of a chiral epoxide using whole cells of Bacillus megaterium ECU1001 in a biphasic system

Optically active epoxides can be prepared by kinetic resolution of racemic mixtures using stereospecific epoxide hydrolases. To increase the bio-resolution efficiency of a sparingly water-soluble epoxide (glycidyl phenyl ether, GPE), we investigated the use of organic/aqueous two-phase system. Various conditions were systematically examined and optimized in shake flasks. Isooctane was found to be the most suitable solvent as the organic phase. The phase volume ratio (ϕ_o/w) and biocatalyst concentration were shown to be sensitive parameters affecting both the reaction rate and the enzyme enantiospecificity in the biphase system. An isooctane/aqueous system was developed to overcome the low solubility and instability of GPE in the aqueous phase, resulting in a significant improvement of enatiomeric ratio (E-value) from 39.5 to 94.0 and an average productivity of 18.8 mg GPE/(h g) biocatalyst to 48.9 mg GPE/(h g) biocatalyst, respectively. Resolution of a 90.1 g/l solution of racemic glycidyl phenyl ether in isooctane phase was successfully carried out in a mechanically stirred reactor (120 ml), affording (S)-glycidyl phenyl ether in high (100%) enantiomeric excess with a yield of 44.5%.     

Optimized synthesis of (Z)-3-hexen-1-yl caproate using germinated rapeseed lipase in organic solvent

(Z)-3-hexen-1-yl esters are important green top-note components of food flavors and fragrances. Effects of various process conditions on (Z)-3-hexen-1-yl caproate synthesis employing germinated rapeseed lipase acetone powder in organic solvent were investigated. Rapeseed lipase catalyzed ester formation more efficiently with non-polar compared to polar solvents despite high enzyme stability in both types of solvents. Maximum ester yield (90%) was obtained when 0.125 M (Z)-3-hexen-1-ol and caproic acid were reacted at 25 °C for 48 h in the presence of 50 g/L enzyme in heptane. Enzyme showed little sensitivity towards a_w with optimum yield at 0.45, while added water did not affect ester yield. Esterification reduced by increasing molecular sieves (>0.0125%, w/v). The highest yields of caproic acid were obtained with isoamyl alcohol (93%) followed by butanol and (Z)-3-hexen-1-o1 (88%) respectively reflecting the enzyme specificity for straight and branched chain alcohols. Secondary alcohols showed low reactivity, while tertiary alcohol had either very low reactivity or not esterified at all. A good relationship has been found between ester synthesis and the solvent polarity (log P value); while no correlation for the effect of solvents on residual enzyme activity was observed. It may be concluded that germinated rapeseed lipase is a promising biocatalyst for the synthesis of valuable green flavor note compound. The enzyme also showed a wide range of temperature stability (5–50 °C).     

Biocatalytic ethanolysis of palm oil for biodiesel production using microcrystalline lipase in tert-butanol system

Biocatalytic synthesis is a promising environmentally friendly process for the production of biodiesel, a sustainable alternative fuel from renewable plant resources. In order to develop an economical heterogeneous biocatalyst, protein-coated microcrystals (PCMCs) were prepared from a commercial enzyme preparation from a recombinant Aspergillus strain expressing Thermomyces lanuginosus lipase and used for synthesis of biodiesel from palm olein by ethanolysis. Reaction parameters, including catalyst loading, temperature, and oil/alcohol molar ratio have been systematically optimized. Addition of tert-butanol was found to markedly increase the biocatalyst activity and stability resulting in improved product yield. Optimized reactions (20%, w/w PCMC-lipase to triacylglycerol and 1:4 fatty acid equivalence/ethanol molar ratio) led to the production of alkyl esters from palm olein at 89.9% yield on molar basis after incubation at 45 °C for 24 h in the presence of tert-butanol at a 1:1 molar ratio to triacylglycerol. Crude palm oil and palm fatty acid distillate were also efficiently converted to biodiesel with 82.1 and 75.5% yield, respectively, with continual dehydration by molecular sieving. Operational stability of PCMC-lipase could be improved by treatment with tert-butanol allowing recycling of the biocatalyst for at least 8 consecutive batches with only slight reduction in activity. This work thus shows a promising approach for biodiesel synthesis with microcrystalline lipase which could be further developed for cost-efficient industrial production of biodiesel.     

A novel organic solvent-stable alkaline protease from organic solvent–tolerant Bacillus licheniformis YP1A

An organic solvent-stable alkaline protease producing bacterium was isolated from the crude oil contaminant soil and identified as Bacillus licheniformis. The enzyme retained more than 95% of its initial activity after pre-incubation at 40 °C for 1 h in the presence of 50% (v/v) organic solvents such as DMSO, DMF, and cyclohexane. The protease was active in a broad range of pH from 8.0 to 12.0 with the optimum pH 9.5. The optimum temperature for this protease activity was 60 °C, and the enzyme remained active after incubation at 50–60 °C for 1 h. This organic solvent-stable protease could be used as a biocatalyst for organic solvent-based enzymatic synthesis.     

Synthesis of octyl-β-d-glucopyranoside catalyzed by Thai rosewood β-glucosidase-displaying Pichia pastoris in an aqueous/organic two-phase system

We explored the ability of a Thai rosewood β-glucosidase-displaying P. pastoris whole-cell biocatalyst (Pp-DCBGL) system to synthesize alkyl β-d-glucosides. The primary investigation centered on the synthesis of octyl-β-d-glucopyranoside (octyl-glu, OG). OG could be synthesized through reverse hydrolysis reaction with very low efficiency. Then, OG was synthesized between BG and octanol by a transglycosylation reaction. In a 2-ml reaction system, OG was synthesized with a conversion rate of 51.1% in 3 h when 5 mg/ml BG was utilized as the glucosyl donor under optimized conditions. And, even after being reused four times, the Pp-DCBGL was relatively stable. Additionally, a 500-ml-scale reaction system was conducted in a 2-L stirred reactor with a conversion rate of 47.5% in 1.5 h. Moreover, the conversion rate did not decrease after the whole-cell catalyst was reused two times. In conclusion, Pp-DCBGL has high reaction efficiency and operational stability, which is a powerful biocatalyst available for industrial synthesis.     

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: Characterization and application to enzymatic biodiesel production

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40–50 °C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50 °C and was maintained even after an incubation of 24-h at 60 °C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50 °C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.     

Pyruvate producing biocatalyst with constitutive NAD-independent lactate dehydrogenases

Production of pyruvate from lactate through biocatalysis is a valuable process for its simple composition of reaction system and convenience of recovery. Biocatalyst with lactate-induced NAD-independent lactate dehydrogenases (iLDHs) can effectively catalyze lactate into pyruvate. To reduce the cost of biocatalyst preparation caused by indispensable lactate addition, the mutants with constitutive iLDH of Pseudomonas sp. XP-M2 were screened. Mutant XP-LM exhibited high iLDHs activities in minimal salt medium with cheap substrate glucose as the carbon source. The biocatalyst (8.2 g dry cell weight l⁻¹) containing 169.8 U l⁻¹ l-iLDH was prepared with 20 g 1⁻¹ glucose. The cost-effective biocatalyst prepared from the mutant XP-LM could efficiently catalyze lactate into pyruvate with high yield (0.961 mol mol⁻¹). Based on the different thermostability of d-iLDH and l-iLDH in the biocatalyst, whole cells of the strain might also have the potential in production of pyruvate and d-lactate from racemic lactate.     

Efficient production of α-arbutin by whole-cell biocatalysis using immobilized hydroquinone as a glucosyl acceptor

The aim of the present study is to develop an efficient and cost-effective method for α-arbutin production by using whole-cell of Xanthomonas maltophilia BT-112 as a biocatalyst. Hydroquinone (HQ), substrate for the bioconversion as glucosyl acceptor, was immobilized on H107 macroporous resin to reduce its toxic effect on the cells, and the optimal reaction conditions for α-arbutin synthesis were investigated. When 350 g/L H107 resin (254.5 mM HQ) and 20 g/L (4.2 U/g) of cells were shaken in 10 mL Na₂HPO₄–KH₂PO₄ buffer (50 mM, pH 6.5) containing 509 mM sucrose at 35 °C with 150 rpm for 48 h, the final yield of α-arbutin reached 65.9 g/L with a conversion yield of 95.2% based on the amount of HQ supplied. The α-arbutin production was 202% higher than that of the control (free HQ) and the cells maintained its full activity for almost six consecutive batch reactions, indicating a potential for reducing production costs. Additionally, the product was one-step isolated and identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the combination of whole cells and immobilized hydroquinone (IMHQ) is a promising approach for economical and industrial-scale production of α-arbutin.     

An organic solvent and thermally stable lipase from Burkholderia ambifaria YCJ01: Purification,characteristics and application for chiral resolution of mandelic acid

A solvent-tolerant bacterium Burkholderia ambifaria YCJ01 was newly isolated by DMSO enrichment of the medium. The lipase from the strain YCJ01 was purified to homogeneity with apparent molecular mass of 34 kDa determined by SDS-PAGE. The purified lipase exhibited maximal activity at a temperature of 60 °C and a pH of 7.5. The lipase was very stable below 55 °C for 7 days (remaining 80.3% initial activity) or at 30 °C for 60 days. PMSF significantly inhibited the lipase activity, while EDTA had no effect on the activity. Strikingly, the lipase showed distinct super-stability to the most tested hydrophilic and hydrophobic solvents (25%, v/v) for 60 days, and different optimal pH in contrast with the alkaline lipase from B. cepacia S31. The lipase demonstrated excellent enantioselective transesterification toward the S-isomer of mandelic acid with a theoretical conversion yield of 50%, ee_p of 99.9% and ee_s of 99.9%, which made it an exploitable biocatalyst for organic synthesis and pharmaceutical industries.     

Scaling-up the synthesis of myristate glucose ester catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst

The novel whole-cell biocatalyst Candida antarctica lipase B displaying-Pichia pastoris (Pp-CALB) is characterized by its low preparation cost and could be an alternative to the commercial immobilized Candida antarctica lipase B (CALB). This study addresses the feasibility of using Pp-CALB in large scale glucose fatty acid esters production. 1,2-O-Isopropylidene-α-d-glucofuranose (IpGlc) was used as the acyl acceptor to overcome the low solubility of glucose in an organic solvent and to avoid the addition of toxic co-solvents. IpGlc significantly improved the Pp-CALB catalyzing esterification efficiency when using long chain fatty acids as the acyl donor. Under the preferred operating conditions (50 °C, 40 g/L molecular sieve dosage and 200 rpm mixing intensity), 60.5% of IpGlc converted to 6-O-myristate-1, 2-O-isopropylidene-α-d-glucofuranose (C14-IpGlc) after a 96-h reaction in a 2-L stirred reactor. In a 5-L pilot scale test, Pp-CALB also showed a similar substrate conversion rate of 55.4% and excellent operational stability. After C14-IpGlc was collected, 70% trifluoroacetic acid was adopted to hydrolyze C14-IpGlc to myristate glucose ester (C14-Glc) with a high yield of 95.3%. In conclusion, Pp-CALB is a powerful biocatalyst available for industrial synthesis, and this study describes an applicable and economical process for the large scale production of myristate glucose ester.     

Lipase immobilization on O-propargyl and O-pentynyl dextrans and its application for the synthesis of click beetle pheromones

Lipase of Rhizopus arrhizus was immobilized on O-propargyl dextran (PgD) and O-pentynyl dextran (PyD). Compared with Lewatit VP OC 1600 cation ion exchange resin, wood shaves, fuller earth, silica and alumina, PgD with degree of substitution (DS) of 0.68 and a surface of 10 m²/g was found to be the most effective immobilization support and an excellent biocatalyst for esterification reactions in organic solvents as the synthesis of click beetle pheromone geranyl octanoate. PyD (DS 0.44) with a surface of 3.3 m²/g was of similar high efficiency. For the enzymatic esterification the optimum concentration of geraniol and octanoic acid was 0.4 mol L^?1 each. The biocatalyst worked the best in hexane at a moisture level of 0.02%. The enzyme could be repeatedly used and conversion dropped from 80% to 70% after four cycles, while reaction rate even increased when repeatedly employed.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).