Subtilisin Carlsberg in complex with sodium dodecyl sulfate is an effective catalyst for the solid phase segment coupling of peptides on polyvinyl alcohol cryogel

Subtilisin Carlsberg (SC) was shown to catalyze the solid phase segment coupling of peptides in complex with sodium dodecyl sulfate (SDS) in an organic medium on Aminosilochrom and polyvinyl alcohol (PVA) cryogel activated with glutaraldehyde or divinylsulfone. Diamines of different lengths with a general formula NH₂-(CH₂) _n -NH₂ (n = 2, 4, and 6) were used as spacers between the PVA cryogel and the peptide. A model reaction of enzymatic attachment of the Dnp-Ala-Ala-Leu-OMe tripeptide to the PVA cryogel was carried out by treatment with the SDS-SC complex in a mixture of anhydrous ethanol and DMSO (7 : 3, v/v) using a tenfold excess of the carboxyl component. The molar enzyme-substrate ratio was 1 : 88. The effect of the method of matrix activation, length of a spacer, and reaction time on the coupling efficiency was studied. Hexamethylenediamine was found to be the most effective spacer for the enzymatic coupling on the PVA cryogel activated with glutaraldehyde (the reaction proceeded with the highest yield of 60%). The reaction efficiency was considerably lower in the case of ethylenediamine and tetramethylenediamine (10 and 15%, respectively). The best results were obtained on the PVA cryogel activated by divinylsulfone with hexamethylenediamine as a spacer. A two-step condensation of tripeptides was carried out on this support. The second step of condensation was shown to proceed better (in 85% yield) in comparison with the first step (37% yield).     

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.     

A kinetic study of the reaction between glutaraldehyde and horseradish peroxidase.

Horseradish peroxidase was reacted with glutaraldehyde under various reaction conditions. The reaction product was, in a second step, bound covalently to aminohexyl groups attached to Sepharose particles. The influence of pH, time and the concentration ratio of enzyme:glutaraldehyde on the reaction was evaluated. A first step reaction with 100-fold molar excess of glutaraldehyde to horseradish peroxidase at pH 9.5 for 2 hr at room temperature results in a high yield of conjugated enzyme with well preserved enzymatic activity.     

Activation of nylon by alkaline glutaraldehyde solution for enzyme immobilisation

Summary Nylon tube was directly activated by alkaline glutaraldehyde solution. PEI was utilised as a spacer molecule. Glucose oxidase was immobilised to the nylon tube after reactivating the spacer molecules with glutaraldehyde. On immobilising glucose oxidase there was more protein binding and higher immobilised enzyme activity when compared to immobilised enzyme tube activated by triethyloxonium salt. The optimal condition for direct glutaraldehyde activation of nylon was incubation with 18.5% (w/v) glutaraldehyde in 0.12M borate pH 9.0 for 15 min at 90 °.     

Factors affecting the production of prednisolone by immobilization of Bacillus pumilus E601 cells in poly(vinyl alcohol) cryogels produced by radiation polymerization

To increase the yield percent of prednisolone from hydrocortisone (cortisol), Bacillus pumilus E601 (a radioresistant microorganism) was incorporated into poly(vinyl alcohol) (PVA) cryogel grafted with hydroxyethyl-methacrylate (HEMA) as a crosslinking agent. The polymer was prepared by a radiation polymerization technique at 20 kGy from Co-60 source. The optimum temperature for the biotransformation of hydrocortisone by free cells, poly(PVA)/HEMA, and poly(PVA)/HEMA /N-isopropylacrylamide (N-IPAAm) was 30 °C. The highest yield % of prednisolone was obtained by immobilization of the cells on poly(PVA/HEMA), the addition of N-IPAAm to poly(PVA/HEMA) protected the immobilized cells from temperatures above 35 °C during the fermentation process. The optimal pH (buffered pH) of the biotransformation of hydrocortisone by immobilized and free cells was 7.0, but the maximum yield of prednisolone (60%) was obtained by immobilized cells in comparison with free cells (42%) also at pH 7.0. The prednisolone yield reached 60–65% with 1,2-propanediol cosolvent containing media and 60–62% in the case of ethanediol cosolvent containing media at 1% (v/v) of both cosolvents. 10 mg/50 ml Tween 80 the medium increased the prednisolone yield by only 1.1-fold compared with the control. The maximum bioconversion efficiency was obtained at a substrate concentration of 20 mg/50 ml medium. Stability studies showed that the immobilized cells can be used for seven times without any significant decrease in activity.     

Glucose condensation by glucoamylase in organic solvents

Summary Oligosaccharides were synthesized through the enzymatic condensation of D-glucose by glucoamylase in water-organic mixtures with high concentrations of two of diethylene glycol diethyl ether or triethylene glycol dimethyl ether. The effect of water content on the yield of reaction was studied; maximum yield was obtained with 10% (v/v) of water in the two systems. Kinetics of synthesis and products composition were different with the two solvents. 37% of glucose were condensed by action of glucoamylase from a reaction medium containing 20 g/L of glucose and 90% (v/v) of diethylene glycol diethyl ether.     

Racemization free coupling of peptide segments. Synthesis of an insect neuropeptide.

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed alpha-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt/NMM always rendered highly optically impure products containing 10-20% of the D-epimer.     

A study on the effects of covered stents on tissue prolapse

Polyvinyl alcohol (PVA) cryogel covered stents may reduce complications from thrombosis and restenosis by decreasing tissue prolapse. Finite element analysis was employed to evaluate the effects of PVA cryogel layers of varying thickness on tissue prolapse and artery wall stress for two common stent geometries and two vessel diameters. Additionally, several PVA cryogel covered stents were fabricated and imaged with an environmental scanning electron microscope. Finite element results showed that covered stents reduced tissue prolapse up to 13% and artery wall stress up to 29% with the size of the reduction depending on the stent geometry, vessel diameter, and PVA cryogel layer thickness. Environmental scanning electron microscope images of expanded covered stents showed the PVA cryogel to completely cover the area between struts without gaps or tears. Overall, this work provides both computational and experimental evidence for the use of PVA cryogels in covered stents.     

The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps. (c) 1996 John Wiley & Sons, Inc.     

Pseudomonas fluorescens lipase immobilization on polysiloxane–polyvinyl alcohol composite chemically modified with epichlorohydrin

The technique based on sol–gel approach was used to generate silica matrices derivatives by hydrolysis of silane compounds. The present work evaluates a hybrid matrix obtained with tetraethoxysilane (TEOS) and polyvinyl alcohol (PVA) on the immobilization yield of lipase from Pseudomonas fluorescens. The resulting polysiloxane–polyvinyl alcohol (POS–PVA) matrix combines the property of PVA as a suitable polymer to retain proteins with an excellent optical, thermal and chemical stability of the host silicon oxide matrix. Aiming to render adequate functional groups to the covalent binding with the enzyme the POS–PVA matrix was chemically modified using epichlorohydrin. The results were compared with immobilized derivative on POS–PVA activated with glutaraldehyde. Immobilization yield based on the recovered lipase activity depended on the activating agent and the highest efficiency (32%) was attained when lipase was immobilized on POS–PVA activated with epichlorohydrin, which, probably, provided more linkage points for the covalent bind of the enzyme on the support. This was confirmed by determining the morphological properties using different techniques as X-ray diffraction and scanning electron microscopy (SEM). Comparative studies were carried out to attain optimal activities for free lipase and immobilized systems. For this purpose, a central composite experimental design with different combinations of pH and temperature was performed. Enzymatic hydrolysis with the immobilized enzyme in the framework of the Michaelis–Menten mechanism was also reported. Under optimum conditions, the immobilized derivative on POS–PVA activated with epichlorohydrin showed to have more affinity for the substrate in the hydrolysis of olive oil, with a Michaelis–Menten constant value (K_m) of 293 mM, compared to the value of 401 mM obtained for the immobilized lipase on support activated with glutaraldehyde. Data generated by DSC showed that both immobilized derivatives have similar thermal stabilities.     

Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin

Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine (phenylglycine amide or phenylglycine methyl ester) and 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional production of 7-ADCA takes place via a chemical ring expansion step and an enzymatic hydrolysis step starting from penicillin G. However, 7-ADCA can also be produced by the enzymatic hydrolysis of adipyl-7-ADCA. In this work, this reaction was combined with the enzymatic synthesis reaction and performed simultaneously (i.e., one-pot synthesis). Furthermore, in situ product removal by adsorption and complexation were investigated as means of preventing enzymatic hydrolysis of cephalexin. We found that adipyl-7-ADCA hydrolysis and cephalexin synthesis could be performed simultaneously. The maximum yield on conversion (reaction) of the combined process was very similar to the yield of the separate processes performed under the same reaction conditions with the enzyme concentrations adjusted correctly. This implied that the number of reaction steps in the cephalexin process could be reduced significantly. The removal of cephalexin by adsorption was not specific enough to be applied in situ. The adsorbents also bound the substrates and therewith caused lower yields. Complexation with beta-naphthol proved to be an effective removal technique; however, it also showed a drawback in that the activity of the cephalexin-synthesizing enzyme was influenced negatively. Complexation with beta-naphthol rendered a 50% higher cephalexin yield and considerably less byproduct formation (reduction of 40%) as compared to cephalexin synthesis only. If adipyl-7-ADCA hydrolysis and cephalexin synthesis were performed simultaneously and in combination with complexation with beta-naphthol, higher cephalexin concentrations also were found. In conclusion, a highly integrated process (two reactions simultaneously combined with in situ product removal) was shown possible, although further optimization is necessary.     

Production of low-lactose milk by means of nonisothermal bioreactors

The effect of the immobilization time on the activity of immobilized beta-galactosidase from K. lactis was investigated. Six biocatalytic membranes, different only for the time of the enzyme immobilization, were obtained by using nylon membranes grafted with glycidyl methacrylate (GMA) and activated by hexamethylenediamine (HMDA) and glutaraldehyde (Glu), used as spacer and coupling agent, respectively. Comparison between the isothermal and nonisothermal yield of these biocatalytic membranes was carried out in the process of lactose hydrolysis in milk. All of the results, reported as a function of the immobilization time, have evidenced the influence of our variable parameter on the activity of the catalytic membranes. The membrane giving highest yield under isothermal and nonisothermal conditions was that obtained with 2 h of immobilization time. The industrial application of these membranes has been discussed in terms of percentage reduction of the production times.     

Immobilization and kinetics of catalase onto magnesium silicate

Bovine liver catalase was immobilized covalently with glutaraldehyde, or glutaraldehyde+3-aminopropionic acid as a spacer, onto magnesium silicate. The coupling time was determined as 2 h for immobilization. The pH and temperature optima as well as the changes in the kinetics (K_m, V_max, E_a) of the immobilized catalase was observed and discussed. Immobilized catalase preparations showed higher storage stabilities than free catalase. The half-life of free catalase, catalase immobilized via glutaraldehyde and catalase immobilized via glutaraldehyde+spacer were calculated as 2, 55 and 10 days at room temperature and 4, 85 and 107 days at 5 °C, respectively. The operational stability of the catalase immobilized via glutaraldehyde was higher than the catalase immobilized via glutaraldehyde+spacer. The remaining activity of the catalase immobilized via glutaraldehyde was about 90% and that of the catalase immobilized via glutaraldeyde+spacer was about 30% after 20 cycles of batch operation.     

高分子膜载体固定化酵母催化2-辛酮不对称还原

以戊二醛交联尼龙6膜载体固定化面包酵母DX213,采用固定化酵母细胞催化2-辛酮不对称还原得到(R)-2-辛醇。系统考察了有机溶剂、反应时间、pH、底物、辅助底物和热处理等因素对反应的产率和光学选择性的影响。结果表明,上述因素对酵母细胞催化不对称合成(R)-2-辛醇反应均有显著影响。二氯甲烷为该反应最适有机溶剂,在固定化细胞57 g/L(50℃预热50 min),水相与有机溶剂相体积比4/1,pH 7.0,初始2-辛酮浓度为60 mmoL/L(分别在反应0,10,17 h等分添加),蔗糖5.7 g/L和28℃条件下反应48 h,(R)-2-辛醇的产率和e.e.值分别达到89.3%和96.8%。     

Immobilization of hydrocarbon-oxidizing bacteria in poly(vinyl alcohol) cryogels hydrophobized using a biosurfactant

A simple biosurfactant-based hydrophobization procedure for poly(vinyl alcohol) (PVA) cryogels was developed allowing effective immobilization of hydrocarbon-oxidizing bacteria. The resulting partially hydrophobized PVA cryogel granules (granule volume 5 microl) contained sufficient number (6.5 x 10(3)) of viable bacterial cells per granule, possessed high mechanical strength and spontaneously located at the interface in water-hydrocarbon system. Such interfacial location of PVA granules allowed high contact of immobilized biocatalyst with hydrophobic substrate and water phase, thus providing bacterial cells with mineral and organic nutrients. As a result, n-hexadecane oxidation efficiency of 51% after 10-day incubation was achieved using immobilized biocatalyst. PVA cryogels with increased hydrophobicity can be used for immobilization of bacterial cultures performing oxidative transformations of water-immiscible organic compounds. Immobilization of in situ biosurfactant producing Rhodococcus bacteria into PVA cryogel is discussed. PVA cryogel granules with entrapped alkanotrophic rhodococcal cells were stable after 10-month storage at room temperature.     

Activity and operational stability of immobilized mandelonitrile lyase in methanol/water mixtures

Summary The enzyme mandelonitrile lyase was covalently immobilized on solid support materials using different methods. Immobilization on porous silica using coupling with glutaraldehyde afforded preparations with high enzyme loading (up to 9% (w/w)). The immobilized enzyme was used in a packed bed reactor for the continuous production of d-mandelonitrile from benzaldehyde and cyanide. The influence of the flow rate, pH, substrate concentrations and enzyme loading on the reaction yield and the enantiomeric purity of the product was investigated. In order to suppress the competing spontaneous reaction, the enzymatic reaction must be rapid. A flow rate of 9.5 ml/min (0.1 M benzaldehyde and 0.3 M HCN) through a 3 ml reactor afforded a 86% yield of mandelonitrile with 92% enantiomeric excess. No leakage of enzyme occurred under continuous operation. One column was used continuously for 200 h without any decrease in yield or enantiomeric purity of the product. High concentrations of benzoic acid were shown to decrease the operational stability of the system.     

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture

The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.     

A two-step, one-pot enzymatic synthesis of ampicillin from penicillin G potassium salt

A two-step, one-pot synthesis of ampicillin from penicillin G potassium salt (PGK) in aqueous buffer/organic co-solvent has been achieved. Ethylene glycol (EG) was chosen as the organic co-solvent. Factors including co-solvent content, enzyme loading, reaction temperature and substrate concentration were investigated. The optimum conditions were as follow: pH 8.0 phosphate buffer solution, 50% EG (v/v), 25 °C, 100 mM PGK and 300 mM d-phenylglycine methyl ester (D-PGM), 43.2 IU/ml IPA-750. The maximum yield was 57.3% after a reaction time of 17 h. It is the first report about the synthesis of ampicillin from penicillin G potassium salt in one-pot combining the enzymatic hydrolysis and the subsequent enzymatic condensation, and the novel methodology will have important application in the β-lactam antibiotics industry.     

Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis using pore matrix crosslinked with glutaraldehyde

The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid was 75% by immobilized system in batch reaction. No activity was lost after 15 cycles and about 65% enzyme activity was retained at the end of the 31th cycle.     

The enzymatic segment condensation of peptides on a solid phase in organic medium

The segment condensation of peptides on a solid phase (Aminosilochrom) in organic medium catalyzed by a subtilisin complex with sodium dodecylsulfate was studied. The dependence of the efficiency of the enzymatic coupling of tripeptides with the basic structure X-Ala-Ala-Y-OMe [where X = Z, Boc, or Dnp and Y = Leu or Glu(OMe)] on the spacer content on the support and on the structure of the acylating component was investigated. The tripeptide segments were successively coupled to Aminosilochrom containing the Met-Ala-Gly spacer, and the peptidylaminosilochroms Dnp-Ala-Ala-Leu-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Dnp-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A (A is the Aminosilochrom residue) were obtained in satisfactory yields. It was shown by these examples that the second and third segments are attached in yields higher than that for the first segment and the coupling efficiency does not depend on the amino acid composition of the acylating component.