Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

Lipase-catalysed synthesis of glucose fatty acid esters in tert-butanol

Synthesis of 6-O-acylate--d-glycopyranose from underivatised substrates in anhydrous tert-butanol was achieved using immobilised lipases from Candida antarctica and Mucor miehei. Except for acetic acid, the initial reaction rates with the C. antarctica lipase were independent of acyl donor chain lengths and in a range of 3.9±0.4 mol glucose converted min–1 g enzyme preparation. The catalytic activity of the M. miehei lipase increased with increasing acyl donor chain length with a maximum for stearic acid of 0.45 mol min–1 g. Using maltose as substrate, the catalytic activity decreased by a factor of 48 and 20 with the lipase from C. antarctica and M. miehei, respectively, while with maltotriose no reaction was observed.     

Esterification of methyl glycoside mixtures by lipase catalysis

The acylation of a mixture of methyl -D-galacto-, methyl -D-gluco- and methyl -D-mannopyranosides by octanoic acid was catalysed by lipases from Candida antarctica, Pseudomonas cepacia, Candida rugosa OF or Mucor miehei in acetonitrile at 45°C. The methyl glycopyranosides had the same anomeric configuration as that of the softwood hemicellulose galactoglucomannan. C. rugosa OF lipase had almost no substrate specificity and P. cepacia lipase had a high substrate specificity for the esterification of methyl -D-galactopyranoside.     

Enzymatic procedures in the preparation of regioprotected D-fructose derivatives

A combination of different lipases from Pseudomonas cepacia, Candida antarctica B, Candida rugosa and Mucor miehei, aided the regioesterification of the free fructose allowing the synthesis of 1,6-di-O-acetyl-D-fructofuranose, 1,4,6-tri-O-acetyl-D-fructofuranose, 1,6-di-O-acetyl-4-O-benzoyl-D-fructofuranose and 1,6-di-O-benzoyl-D-fructofuranose. Using C. antarctica B and C. rugosa lipases the alcoholysis of fructose peracetate (alpha, beta-form) has furnished 1,2,3,4-tetra-O-acetyl-alpha-D-fructofuranose and 2,3,4,6-tetra-O-acetyl-beta-D-fructofuranose. 1,4,6-Tri-O-acetyl-D-fructofuranose was successfully employed to produce a rare ketohexose, namely D-psicose.     

Selectivity of Rhizomucor miehei lipase as affected by choice of cosubstrate system in ester modification reactions in organic media

Fatty acid (FA) selectivity of immobilized Rhizomucor miehei lipase was determined for various cosubstrate systems for ester modification involving competing n-acyl-donor substrates of even-chain length (C4-C16; FA or their methyl esters, FAME) and either n-propanol or propyl acetate in hexane. Acyl-chain-length optima were observed for C8 and C14/16 in all cases. Upon changing between cosubstrate systems of [FA + propanol] to [FAME + propanol] to [FAME + propyl acetate], there was a general shift in selectivity toward shorter-chain-length FA (C4-C8). The greatest degree of reaction selectivity (based on ratios of selectivity constants) among the FA substrates was 3.1 for the [FA + propanol], 2.5 for the [FAME + propanol], and 1.4 for the [FAME + propyl acetate] cosubstrate systems. For esterification reactions between C6 FA and reactive members of a series of aliphatic and aromatic alcohols, the greatest degree of selectivity observed was 3.6.     

Improved 1,3-propanediol production with hemicellulosic hydrolysates (corn straw) as cosubstrate: Impact of degradation products on Klebsiella pneumoniae growth and 1,3-propanediol fermentation

To improve 1,3-propanediol (1,3-PD) production by an economic and efficient approach, hemicellulosic hydrolysates (HH) used as cosubstrate resulted in more biomass and higher reducing power for 1,3-PD production. The effects of primary degradation products such as individual sugars (xylose, glucose, mannose, arabinose and galactose) and major inhibitors (furfural, acetate and formate) on the Klebsiella pneumoiae growth and 1,3-PD production were investigated in this study. Xylose and mannose could efficiently promote the 1,3-PD production and cell growth. Furfural (0.28 g/l) and sodium acetate (1.46 g/l) in low concentration were not inhibitory to Klebsiella pneumoniae, rather they have stimulatory effect on the growth and 1,3-PD biosynthesis, especially the acetate. In fed-batch fermentation with HH as cosubstrate, the final 1,3-PD production, conversion from glycerol and productivity were 71.58 g/l, 0.65 mol/mol and 1.93 g/l/h, respectively, which were 17.8%, 25.0% and 17.7% higher than that from glycerol alone.     

Microwave-assisted rapid characterization of lipase selectivities

A rapid screening procedure for characterization of lipase selectivities using microwaves was developed. The rate of reaction of various commercial lipases (porcine pancreas, Mucor miehei, Candida rugosa, Pseudomonas cepacia) as well as lipases from laboratory isolates-Bacillus stearothermophilus and Burkholderia cepacia RGP-10 for triolein hydrolysis was 7- to 12-fold higher in a microwave oven as compared to that by pH stat. The esterification of sucrose/methanol and ascorbic acid with different fatty acids was also achieved within 30 s in a microwave using porcine pancreas, B. stearothermophilus SB-1 and B. cepacia RGP-10 lipases. The relative rates and selectivity of the lipases both for hydrolytic and synthesis reactions remains unaltered. However, the rate of reaction was dynamically enhanced when exposed to microwaves. Microwave-assisted enzyme catalysis can become an attractive procedure for rapid characterization of large number of enzyme samples and substrates, which otherwise is a cumbersome and time-consuming exercise.     

Enzymatic synthesis of alpha-butylglucoside linoleate in a packed bed reactor for future pilot scale-up

The enzymatic synthesis of a mixture of unsaturated fatty acid alpha-butylglucoside esters, containing more than 60% alpha-butylglucoside linoleate, was achieved through lipase-catalyzed esterification. The continuous evaporation under reduced pressure of the water produced enabled substrate conversions greater than 95% to be reached. Two immobilized lipases from Candida antarctica (Chirazyme L2, c.-f., C2) and Rhizomucor miehei (Chirazyme L9, c.-f.) were compared in stirred batch and packed bed configurations. When the synthesis was carried out in stirred batch mode, C. antarctica lipase appeared to be of greater interest than the R. miehei enzyme in terms of stability and regioselectivity. Surprisingly, a change in the process design to a packed bed configuration enabled the stability of R. miehei lipase to be significantly improved, while the C. antarctica lipase efficiency to synthesize unsaturated fatty acid alpha-butylglucoside esters was slightly decreased. Water content in the microenvironment of the biocatalyst was assumed to be responsible for such changes. When the process is run in stirred batch mode, the conditions used promote the evaporation of the essential water surrounding the enzyme, which probably leads to R. miehei lipase dehydration. In contrast, the packed bed design enabled such water evaporation in the microenvironment of the biocatalyt to be avoided, which resulted in a tremendous improvement of R. miehei lipase stability. However, C. antarctica lipase led to the formation of 3% diesters, whereas the final percentage of diesters reached 21% when R. miehei enzyme was used as biocatalyst. A low content of diesters is of greater interest in terms of alpha-butylglucoside linoleate application as linoleic acid carrier, and therefore the enzyme choice will have to be made depending on the properties expected for the final product.     

耐高浓度甘油的1,3-丙二醇产生菌诱变选育与固定化培养初步研究

紫外诱变筛选耐高浓度甘油的1,3-PD克雷伯杆菌,诱变条件为30 W紫外灯,距离34 cm,照射6 m in,平板涂布稀释度为10-5,经六轮诱变筛选出耐90 g/L甘油的变异菌株,甘油转化率稳定在45%左右,1,3-PD生成量稳定在40 g/L左右,比原始出发菌株提高了近30%,诱变菌株经考察具有一定的遗传稳定性。在此基础上应用溶胶-凝胶法进行固定化细胞实验并与游离细胞进行了四个批次的对比连续发酵,结果显示溶胶-凝胶法具有一定的稳定性。     

Lipase-mediated hydrolysis of blackcurrant oil

Four commercially available lipases, both free and immobilized, were tested for their ability to catalyze hydrolysis of blackcurrant (Ribes nigrum) oil using two different approaches. The lipase from Mucor miehei was studied free and immobilized in two different ways. The former series of enzymic reactions were performed in tap water at 40 degrees C, but the latter series of enzymic processes were carried out in mixtures of isooctane and phosphate buffer (in a typical 2/1 ratio of the components) at 30 degrees C. These conditions were optimized to increase and/or to maximize the yields of the products, which were priority targets in this study. A rate of hydrolysis and a selective preference of the hydrolytic enzymes towards fatty acids, with a special focus on enrichment of alpha-linolenic acid and/or gamma-linolenic acid, were studied. Higher rates of hydrolysis of the blackcurrant oil in the former series of reactions were observed with the immobilized lipase from Pseudomonas cepacia used as biocatalyst. In the latter approach, the most favorable results of the rate of hydrolysis of the target blackcurrant oil were achieved with the immobilized lipase from Mucor miehei employed as biocatalyst. Only three lipases, selected from a series of lipases tested during this investigation, displayed specificity towards alpha-linolenic acid and gamma-linolenic acid, i.e. the immobilized lipase from P. cepacia, lipase from M. miehei and lipase from P. fluorescens.     

基于甘油的1,3-丙二醇生物合成的代谢局限及其改造策略

粗甘油是生物柴油生产中的主要副产物,一些微生物可将甘油转化为重要化工原料1,3-丙二醇(1,3-PD),而利用这些微生物野生菌株生物合成1,3-PD会存在一些局限性,如底物抑制、产物抑制等。文中从1,3-丙二醇的甘油生物转化途径与这些局限性出发,总结了生物合成中存在的问题,并针对这些问题提出了一些基于基因敲除或基因过表达等基因工程技术的改造方法,综述了利用基因工程菌生物转化甘油生成1,3-丙二醇的最新研究进展。     

Enrichment of butteroil with conjugated linoleic acid via enzymatic interesterification (acidolysis) reactions

Microbial lipases from Candida cylindracea, Pseudomonas sp., Mucor miehei and Candida antarctica were screened for their ability to incorporate conjugated linoleic acid into butteroil triacylglycerides. The lipase from Candida antarctica was employed in a substrates-only medium to increase the conjugated linoleic acid content of the acylglycerides from 0.6 to 15 g/100 g fat.     

Biodiesel production from triolein and short chain alcohols through biocatalysis

Oleic acid alkyl esters (biodiesel) were synthesised by biocatalysis in solvent-free conditions. Different commercial immobilised lipases, namely Candida antarctica B, Rizhomucor miehei, and Pseudomonas cepacia, were tested towards the reaction between triolein and butanol to produce butyl oleate. Pseudomonas cepacia lipase resulted to be the most active enzyme reaching 100% of conversion after 6h. Different operative conditions such as reaction temperature, water activity, and reagent stoichiometric ratio were investigated and optimised. These conditions were then used to investigate the effect of linear and branched short chain alcohols. Methanol and 2-butanol were the worst alcohols: the former, probably, due to its low miscibility with the oil and the latter because secondary alcohols usually are less reactive than primary alcohols. Conversely, linear and branched primary alcohols with short alkyl chains (C(2)--C(4)) showed high reaction rate and conversion. A mixture of linear and branched short chain alcohols that mimics the residual of ethanol distillation (fusel oil) was successfully used for oleic acid ester synthesis. These compounds are important in biodiesel mixtures since they improve low temperature properties.     

Construction and Characterization of a 1,3-Propanediol Operon

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B₁₂, which must be provided in E. coli fermentations. However, the amount of coenzyme B₁₂ needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.     

Optimization of culture conditions for 1,3-propanediol production from crude glycerol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> using response surface methodology

To produce 1,3-propanediol (1,3-PD) from crude glycerol, cultivation conditions were optimized by response surface methodology (RSM) based on a 2⁵ factorial central composite design (CCD). RSM was adopted to derive a statistical model for the individual and interactive effects of crude glycerol, (NH₄)₂SO₄, pH, cultivation time and temperature on the production of 1,3-PD. Optimal conditions for maximum 1,3-PD production were as follows: crude glycerol, 35 g/L; (NH₄)₂SO₄, 8 g/L; pH, 7.37; cultivation time, 10.8 h; temperature, 36.88°C. Under these optimal conditions, the design expert presented the maximal numerical solution with a predicted 1,3-PD production level of up to 13.74 g/L. The experimental production of 1,3-PD yielded 13.8 g/L, which was in close agreement with the model prediction.     

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.     

Characterization of 1,3-propanediol oxidoreductase (DhaT) from <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> J2B

1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k _cat , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K _m , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD⁺) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag⁺ and Hg²⁺. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.     

Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol

In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.     

Genome Shuffling in Clostridium diolis DSM 15410 for Improved 1,3-Propanediol Production

Several microorganisms are known for their efficient anaerobic conversion of glycerol to 1,3-propanediol, with Clostridium diolis DSM 15410 as one of the better performers in terms of molar yield and volumetric productivity. However, this performance is still insufficient to compete with established chemical processes. Previous studies have shown that high concentrations of 1,3-propanediol, glycerol, and fermentation side products can limit the productivity of C. diolis DSM 15410. Here, we describe the use of genome shuffling for improved 1,3-propanediol fermentation by the strict anaerobe C. diolis DSM 15410. By using chemical mutagenesis, strains with superior substrate and product tolerance levels were isolated and higher product yields were obtained. These superior strains were then used for genome shuffling and selection for 1,3-propanediol and organic acid tolerance. After four rounds of genome shuffling and selection, significant improvements were observed, with one strain attaining a 1,3-propanediol volumetric yield of 85 g/liter. This result represents an 80% improvement compared to the yield from the parental wild-type strain.The use of biomass instead of petrochemical feedstock could facilitate the sustainable production of many chemicals, but this approach has proven economically feasible in only a few cases (4, 17, 20, 21). The microbial production of 1,3-propanediol (1,3-PD) provides an interesting case study because this monomer is used to produce several plastics, including the relatively new and highly versatile polytrimethylene terephthalate, which has significantly increased demand for 1,3-PD (12, 23, 24). Polytrimethylene terephthalate is currently produced from petrochemical feedstock in a process that involves the conversion of ethylene oxide into 3-hydroxypropionaldehyde by hydroformylation under high pressure and then further into 1,3-PD by hydrogenation using a nickel or rubidium catalyst (21).Several companies have investigated the sustainable production of 1,3-PD from biomass. For example, DuPont and Genencor transferred the relevant 1,3-PD biosynthetic genes from Klebsiella pneumonia into Escherichia coli and further modified carbohydrate metabolism and transport so that 1,3-PD could be synthesized from glucose (10, 11; M. Emptage, S. L. Haynie, L. A. Laffend, J. Pucci, and G. M. Whited, 2001, Patent Cooperation Treaty international application WO 2001/01/12833). Large-scale production of 1,3-PD by this approach is likely to be too expensive due to the high input costs of vitamin B₁₂ and antibiotics, so the use of glycerol as an alternative feedstock has been investigated, although this strategy requires additional enzymes and a shift from aerobic to anaerobic conditions (15, 25, 30).Clostridium diolis DSM 15410 (formerly C. butyricum DSM 5431) can produce 1,3-PD from glycerol under anaerobic conditions and is therefore a desirable alternative to E. coli given the relative costs of industrial aerobic and anaerobic fermentation (6, 8, 9, 22). However, the efficiency of conversion is not yet high enough for an industrial process. The production of 1,3-PD by C. diolis is limited by inhibition from both substrates and products, as well as organic acids produced as fermentation by-products (13).Classical strain improvement has significantly increased 1,3-PD production, but this is a slow process and the mutations are predominantly neutral or detrimental (2). We have therefore approached the problem using genome shuffling, which is more efficient and reliable for engineering complex phenotypes, as demonstrated in several other examples of microbial strain development (16, 26, 31). Genome shuffling offers the advantages of accumulated beneficial mutations and the removal of unnecessary mutations due to simultaneous changes at different positions throughout the genome and, therefore, yields microbes of superior fitness (29). We applied both the classical approach and genome shuffling to C. diolis DSM 15410 to improve the production of 1,3-PD, which is a necessary prerequisite for the fermentation process. To our knowledge, this study is the first example of genome shuffling in a strictly anaerobic microorganism.     

Molecular Cloning, Co-expression, and Characterization of Glycerol Dehydratase and 1,3-Propanediol Dehydrogenase from Citrobacter freundii

1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.