Peptide synthesis with immobilized carboxypeptidase Y

Enzymatic peptide synthesis was investigated using carboxypeptidase Y immobilized with glutaraldehyde on 10 mum microparticulate amino-silica. Carboxypeptidase Y was immobilized with 98.5% recovery of active enzyme to yield the immobilized enzyme having 0.55 units esterase activity/mg amino-silica support. The stability of the immobilized enzyme was examined as a function of pH, temperature, and reactant concentrations. Immobilized Carboxypeptidase Y was used in stirred batch and recirculating packed-bed reactors for peptide synthesis. Packed-bed reactors (40 x 4.6 mm, 60 x 4.6 mm) were used to catalyze the synthesis of 170 mg N-benzoyl-L-arginyl-L-methioninamide, 380 mg N-benzoyl-L-arginyl-L-methionyl-L-leucinamide, and 200 mg N-benzoyl-L-arginyl-L-methionyl-L-leucyl-L-phenylalaninamide in 8, 3, and 1 hour, respectively, as intermediates in the synthesis of L-methionyl-L-leucyl-L-phenylalanine. No inactivation of the immobilized enzyme was observed during the course of the reactions. The N-benzoyl-L-arginyl group served to increase the water solubility of the peptides and was removed by immobilized trypsin at the end of synthesis to obtain the final product. While the first two syntheses were conducted with aqueous reaction mixtures, the synthesis of N-benzoyl-L-arginyl-L-methionyl-L-leucyl-L-phenylalaninamide was carried out in a reaction mixture containing dimethylformamide to avoid precipitation of the product. HPLC and amino acid analysis confirmed the high purity and amino acid composition of the final product.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis

Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and purity. These features make enzyme-immobilized MNPs applicable to organic synthesis.     

Cross-linked aggregates of penicillin acylase: robust catalysts for the synthesis of β-lactam antibiotics

A novel method for the immobilization of penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11) is reported. It involves the physical aggregation of the enzyme, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs). Compared with conventionally immobilized penicillin G acylases, these CLEAs possess a high specific activity as well as a high productivity and synthesis/hydrolysis (S/H) ratio in the synthesis of semi-synthetic antibiotics in aqueous media. Moreover, they are active in a broad range of polar and apolar organic solvents.     

Gluco-oligosaccharide synthesis by free and immobilized beta-glucosidase

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. (c) 1993 John Wiley & Sons, Inc.     

Expression of secreted His-tagged S-adenosylmethionine synthetase in the methylotrophic yeast Pichia pastoris and its characterization, one-step purification, and immobilization

S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cerevisiae was successfully produced at high level ( approximately 200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His6-tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 degrees C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl-Met and ATP.     

Evaluation of the performance of immobilized penicillin G acylase using active-site titration

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.     

Preparation and properties of soluble-insoluble immobilized proteases

In order to carry out an effective enzyme reaction, the preparation of soluble-insoluble immobilized enzyme was investigated. Proteases were selected as model enzymes, and their immobilization was carried out by using an enteric coating polymer as a carrier. Among the polymers tested, methacrylic acid-methylacrylate-methylmethacrylate copolymer (MPM-06) gave the most active soluble-insoluble immobilized papain. This immobilized papain showed insoluble from below pH 4.8 and soluble form above pH 5.8; it was also soluble in water-miscible organic solvent. It was reusable and more stable with heat and water-miscible organic solvents than native proteases. Furthermore, various proteases could be immobilized by using MPM-06 with high activity. Chymotrypsin immobilized by this method catalyzed the effective peptide synthesis in a heterogeneous reaction system containing water-miscible organic solvent.     

Peptide synthesis in aqueous-organic solvent mixtures with alpha-chymotrypsin immobilized to tresyl chloride-activated agarose

alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.     

His-tagged Horse Liver Alcohol Dehydrogenase: Immobilization and application in the bio-based enantioselective synthesis of (S)-arylpropanols

The novel histidine-tagged Horse Liver Alcohol Dehydrogenase (His-HLADH-EE) was successfully purified and covalently immobilized onto a solid support in a one-step procedure through a metal-directed technique. A full characterization of the immobilized enzyme was carried out. Effects of pH, temperature and organic co-solvents were deeply investigated and they showed a shift in the optimum pH with respect to the free form as well as increased stability to temperature and solvents. The immobilized His-HLADH-EE proved to be effective as catalyst in the reduction of aliphatic and aromatic aldehydes. Application of the free and immobilized His-HLADH-EE to the chemo-enzymatic synthesis of (S)-Profenols demonstrated enhanced enantioselectivity and high reusability of the immobilized form. The achievement of a robust and effective immobilization of an alcohol dehydrogenase substantiated the use of biocatalytic reduction in the synthesis of primary alcohols and valuable chiral intermediates especially for pharmaceutical industries.     

Synthesis of esters using a nylon-immobilized lipase in batch and continuous reactors.

Direct esterifications using a nylon-immobilized lipase from Candida cylindracea were carried out in batch and continuous-flow reactors. The immobilized enzyme was effective in catalyzing the synthesis of ethylpropionate, isoamylpropionate, and isoamylbutyrate. With ethanol dissolved in hexane as a substrate, the maximum initial esterification rate was 0.02 mole/(h x g of immobilized protein), but the enzyme was stable only when the substrate concentrations were lower than 0.2 M. With isoamyl alcohol in hexane as a substrate, esterification rates as high as 0.085 mole/(h x g of immobilized protein) were observed and the immobilized enzyme was stable over a much broader concentration range. However, in this case, the use of a solvent, such as hexane, was not necessary for esterification, and the enzyme could be employed in equimolar acid/alcohol mixtures. A packed-bed reactor was operated successfully for the continuous synthesis of esters. The reactor was stable for long periods of time, and the steady-state performance could be accurately predicted on the basis of batch reaction experiments.     

Immobilized Candida antarctica lipase B: Hydration, stripping off and application in ring opening polyester synthesis

This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.     

Potential applications of an alcohol-aldehyde/ketone oxidoreductase from thermophilic bacteria

Practical uses of a novel alcohol dehydrogenase from Thermoanaerobium brockii have been examined in crude and purified form. Stoichiometric reduction of NADP (50 mg) was demonstrated with agarose-immobilized enzyme and 0.3 (v/v) 2-propanol solution as reductant. A coenzyme recycle number of 20000 was achieved in enzymatic reactions that employed the alcohol dehydrogenase for NADPH/NADP regeneration. Gram-scale synthesis of chiral R(+) 2-pentanol was shown in a system composed of enzyme, 2-pentanone and 2-propanol as reductant. The effect of temperature, reaction time and substrate concentration on alcohol optical purity was examined. An optical purity of 80% was achieved in the enzymatic synthesis of R(+) 2-pentanol. The enzyme was easily immobilized and stable on an enzyme electrode for analytical detection of alcohols and carbonyls. T. brockii enzyme has potential applications as a commercial alcohol dehydrogenase because of broad substrate specificity and activity at high temperature or high solvent concentration, rare carbonyl si-face stereo-specificity in hydrogen transfer, and high stability and activation of immobilized enzyme.     

纳米材料固定化酶的研究进展

近年来,纳米技术为酶固定化提供了多种纳米级材料,纳米材料固定化酶不仅具有高的酶负载量,而且具有良好的酶稳定性。本文基于纳米材料固定化酶,对纳米材料的种类进行了总结,分析了纳米材料对固定化酶性能的影响,并介绍了纳米级固定化方法及纳米材料固定化酶在生物转化、生物传感器、生物燃料电池等领域的应用。     

Isomaltose formation by free and immobilized dextransucrase

The acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL-B512F with glucose as acceptor is of technical interest for isomaltooligosaccharide (IMOs) synthesis. Different experimental conditions were investigated for free and immobilized enzyme. The data for oligosaccharide formation up to a degree of polymerization 4 were correlated with a model developed earlier, and optimal reaction conditions for immobilized dextransucrase design and application were identified for later continuous application. Furthermore, stability was investigated for free and immobilized enzyme including stabilization by sugars.     

尿苷二磷酸糖固定化酶合成法研究

利用生物酶进行体外催化反应合成不同种类的尿苷二磷酸糖（uridine diphosphate sugar,UDP-糖）,生物酶的重复利用率较低。为提高尿苷二磷酸糖的合成效率及增加产物种类,以镍螯合聚丙烯酸酯树脂为载体,对带有HIS标签的N-乙酰己糖胺激酶（N-acetylhexosamine kinase,NahK）和尿苷转移酶（uridine transferase,GlmU）进行固定化。以固定化NahK和固定化GlmU为催化酶,不同单糖作为底物,研究尿苷二磷酸糖的一锅法合成情况。利用Q柱对产物进行纯化,通过高效液相色谱法、质谱法、核磁共振氢谱法对反应产物进行检测。确定了镍螯合聚丙烯酸酯树脂对游离NahK和GlmU的实际载量分别为10和20 mg·g^-1。固定化酶量的最优配比为5.5 g固定化NahK和2.5 g固定化GlmU。固定化酶的最适pH和温度分别为8.0和35℃,且能在重复反应中稳定反应5个批次。葡萄糖、N-乙酰氨基葡萄糖和甘露糖可以参与一锅法反应,生成UDP-糖的相对分子质量分别为566、607、566,而葡萄糖醛酸、半乳糖和果糖在该体系下不能合成相应的UDP-糖。基于固定化酶技术,一锅法可合成UDP-葡萄糖、UDP-N-乙酰氨基葡萄糖、UDP-甘露糖。     

Renewable enzyme reactors based on beds of artificial gel antibodies

A novel approach is described for the synthesis of beds for enzyme reactors. The method is based on the use of artificial antibodies in the form of polyacrylamide gel particles with diameters around 0.1–0.3 mm. These gel particles mimic protein antibodies, raised in experimental animals, in the sense that they selectively recognize and adsorb only the protein present during the preparation of the “antibodies”. The gel antibodies have several advantages over conventional protein antibodies, which can be taken advantage of in the design of enzyme reactors; for instance, if upon prolonged use the immobilized enzyme loses its activity it can easily be replaced by an active enzyme, which is not possible when the enzyme is immobilized via a conventional protein antibody (a new bed with immobilized protein antibodies must be prepared); and equally or more remarkable: the enzyme can be applied in the form of a non-purified extract since the selectivity of the artificial gel antibodies is so high that they will “fish-out” the enzyme, but no other proteins in the extract. In addition, no preconcentration of the enzyme solution is required prior to the immobilization, since the enzyme is enriched at the top of the column upon the application. These unique properties make enzyme reactors based on artificial gel antibodies very attractive, also in process chromatography. The potential application range of the artificial gel antibodies is enormous since the same method for their synthesis can be used independent of the structure and the size of the “antigen”; for instance, renewable biosensors based on gel antibodies for the selective detection of protein biomarkers, as well as pathogenic viruses, bacteria, and spores (for instance Anthrax) should not be difficult to design.     

Continuous production of maltotetraose using a dual immobilized enzyme system of maltotetraose-forming amylase and pullulanase

In order to produce a product with a high content of maltotetraose, dual-enzyme systems composed of immobilized maltotetraose-forming amylase (G(4)-forming amylase) and pullulanase were studied. The thermostability of individually immobilized enzymes was examined in continuous operation; studies revealed that the enzyme immobilized on "Chitopearl" was much more stable than that immobilized on Diaion HP-50. The effects of operating conditions on the stability of G(4) forming amylase immobilized on "Chitopearl" were examined to confirm that the apparent half-life data could be arranged using the immobilized enzyme stability factor, f(s). As for the dual immobilized enzyme system, six methods of usage were considered, with five yielding a 7-10% (w/w) higher content of maltotetraose product than the single-enzyme system. The effects of operating conditions on the maltotetraose production reaction were examined to confirm that the maltotetraose content of the products could be analyzed using the specific space velocity,SSV. In dual immobilized enzyme systems, pullulanase immobilized on the same carrier as the G(4)-forming amylase was found to be more stable than pullulanase immobilized on separate carriers. The effectiveness of using immobilized pullulanase along with the G(4)-forming amylase was confirmed from constant-conversion operations in which the maltotetraose content in the product was kept at 50% (w/w) in laboratory-scale experimentation.     

The requirement for a primer in the in vitro synthesis of polysaccharide by sweet-corn (1 → 4)-α-d-glucan syntrase

A mixture of (1 → 4)-α-d-glucan synthases was partially purified from sweet corn. The synthesis of polysaccharide from ADP-d-glucose by the enzyme preparation was dependent on added carbohydrate primer in solutions of low ionic strength, but displayed the phenomenon of being apparently primer-independent at high ionic strength in citrate buffer. This phenomenon was further investigated; treatment of the enzyme preparation with immobilized amylases led to the abolition of the apparently unprimed synthesis. The amylase-treated preparation then showed a normal dependence on (1 → 4)-α-d-glucan primer, branched primers being the most effective. The affinity of the enzyme for a branched primer appeared to be enhanced in the presence of citrate. The polysaccharide product of the unprimed reaction was glycogen-like, having an average chain-length of 14. These studies suggest that the phenomenon of unprimed synthesis in “high salt” is explicable in terms of an enhanced affinity of the enzyme for traces of primer in the enzyme preparation, and not to a “de novo” synthesis of polysaccharide, that occurs in the absence of a primer.