The separation of pectinlyase from β-glucosidase in a commercial preparation

This paper describes a new, simple and inexpensive procedure for separating pectinlyase (PL, EC 4.2.2.3) from β-glucosidase (β-glu, EC 3.2.1.21), both of which have potential for use in the beverage processing industry. The method described here, which entails the treatment of crude preparations with bentonite (4% (w/v)) and the acidification of the resulting supernatant to pH 3·5, leads to the production of two enzymic solutions which contain PL and β-glu, respectively. In both solutions the amount of brown pigment is considerably less than in the crude mixture, and partial purification from extraneous proteins is also achieved.     

Stabilisation of β-glucosidase entrapped in alginate and polyacrylamide gels towards thermal and proteolytic deactivation

β-D -Glucosidase was immobilised by entrapment in two different matrices (calcium alginate and polyacrylamide gels), in order to compare how the immobilisation could stabilise the enzyme towards thermal and proteolytic deactivation. While the enzyme trapped in polyacrylamide gel showed an optimum temperature for activity at 10°C lower than that of the free enzyme, the optimal temperature after immobilisation in alginate beads was not altered (60°C). The immobilisation of enzyme in alginate beads caused a larger increase in the thermal stability than the entrapment in polyacrylamide gels. The stabilisation factors obtained as 55, 60 and 65°C for β-glucosidase immobilised in alginate and polyacrylamide gels were 2·03, 3·06, 2·19 and 2·04, 0·35, 1·01, respectively. In contrast, the β-glucosidase immobilised in polyacrylamide gels was more resist-ant in proteolysis than that trapped in alginate beads. © 1998 Society of Chemical Industry     

Production of fructooligosaccharides by β‐fructofuranosidase from Aspergillus sp 27H

β‐fructofuranosidase (EC 3.2.1.26) from Aspergillus sp 27H isolated from soil was investigated for production of fructooligosaccharides (FOS) using whole cells. It possesses hydrolytic and transfructosylating activities that can be altered by modifying the reaction conditions. The optimal conditions for the transfructosylating activity occur in the pH range 5.5–6.0 and at 60 °C, while hydrolytic activity was highest at pH 4.0 and 55 °C. At low sucrose concentration (10 g dm^?3) there was rapid conversion of sucrose to glucose and fructose and very low concentrations of FOS were obtained. However, at sucrose concentrations higher than 216 g dm^?3 the concentrations of hydrolysis products were reduced. Under the following conditions: pH 5.5, temperature 40 °C, sucrose concentration 615 g dm^?3 and enzyme concentration 20β‐fructofuranosidase units g^?1 of sucrose, the FOS concentration reached a maximum value of 376 g dm^?3 (234 g dm^?3 1‐kestose and 142 g dm^?3 nystose) and the proportion of FOS in the solids in the reaction mixture was 600–620 g kg^?1 at 6 h. These results suggest that β‐fructofuranosidase from Aspergillus sp 27H could be an appropriate enzyme for the commercial production of FOS. Copyright © 2004 Society of Chemical Industry     

Studies on the β-glucosidase and endo-1, 4-glucanase activities from Clostridium papyrosolvens

Activities of endo-β-4-glucanase and β-glucosidase from Clostridium papyrosolvens (CECT 747) were studied under different conditions. No indications were found for the presence of more than one β-glucosidase after ion exchange and ammonium sulphate fractionation. β-Glucosidase showed activity against p-nitrophenyl-β-D-glucopyranoside and cellobiose but was not inhibited by δ-gluconolactone. Independent cellobiose phosphorylase activity could also be detected. The β-glucosidase was essentially intracellular. By the use of stationary phase culture assays, enzyme induction at low concentrations of cellobiose was observed, whilst glucose had no effect. Regulation of endo-β-1,4-glucanase and β-glucosidase were compared.     

Production of β‐galactosidase from recombinant Saccharomyces cerevisiae grown on lactose

Improved productivity and costs reduction in fermentation processes may be attained by using flocculating cell cultures. The production of extracellular heterologous β‐galactosidase by recombinant flocculating Saccharomyces cerevisiae cells, expressing the lacA gene (coding for β‐galactosidase) of Aspergillus niger under the ADHI promotor and terminator in a bioreactor was studied. The effects of lactose concentration and yeast extract concentration on β‐galactosidase production in a semi‐synthetic medium were analysed. The extracellular β‐galactosidase activity increased linearly with increasing initial lactose concentrations (5–150 g dm^?3). β‐Galactosidase production also increased with increased yeast extract concentration. During the entire fermentation, no accumulation of the hydrolysed sugars, glucose and galactose, was observed. The catabolic repression of the recombinant strain when cultured in a medium containing equal amounts of glucose and galactose was confirmed. In complete anaerobiosis, the fermentation of lactose resulted in a very slow fermentation pattern with lower levels of β‐galactosidase activity. The bioreactor operation together with optimisation of culture conditions (lactose and yeast extract concentration) led to a 21‐fold increase in the extracellular β‐galactosidase activity produced when compared with preliminary Erlenmeyer fermentations. Copyright © 2004 Society of Chemical Industry     

Ruthenium(II)‐Catalyzed Protocol for Preparation of Diverse α,β‐ and β,β‐Dihaloenones from Diazodicarbonyls

Efficient one‐step syntheses of α,β‐ and β,β‐dihaloenones were achieved by ruthenium(II)‐catalyzed reactions between cyclic or acyclic diazodicarbonyl compounds and oxalyl chloride or oxalyl bromide in moderate to good yields. This methodology offers several significant advantages, which include ease of handling, mild reaction conditions, one‐step reaction, and the use of an effective and non‐toxic catalyst. The synthesized compounds were further transformed into highly functionalized novel molecules bearing aromatic rings on the enone moiety using the Suzuki reaction.

     

Production and properties of Aspergillus niger inulinase

An inulinase producing Aspergillus niger strain was isolated from Compositae rhizosphere soil samples. High inulinase levels were produced on a corn steep liquor (CSL)-maltose medium in the absence of inulin at 28°C within 110 h of fermentation. Media based on CSL-sucrose yielded high cell-bound inulinase activity; on inulin-based media the enzyme was mainly extracellularly produced. Both crude extra-and intracellular inulinase preparations displayed identical pH and temperature optima with maximal activity at pH 4.3–4.4 and at 55–56°C. These properties are favourable in view of large scale inulinase application for pure fructose production. High operation temperatures would avoid microbial contamination of reactors and would allow the use of high inulin-substrate concentrations, a limiting factor in obtaining high conversion ratios. The remarkably low pH optimum prevents colour formation and undesirable chemical side reactions. An advantageous low ratio of invertase to inulinase activity (S/I value) of 0.85 was found for the crude extracellular enzyme preparation. Crude inulin (chicory) extracts are hydrolysed faster than pure inulin. Apart from inulin (100% hydrolysis), sucrose (45%) and raffinose (20%) are also hydrolysed, and no liberation of oligomers or of sucrose from inulin was observed. These facts indicate that the A. niger enzyme is an exo-acting inulinase. The above characteristics make this A. niger inulinase an industrially attractive enzyme for the preparation of pure fructose from inulin-containing agricultural crops.     

The use of covalently immobilized mycelia to modify the β-glucanase system of Aspergillus vesicolor

Aspergillus vesicolor mycelial cells were covalently immobilized on a glutaraldehyde-ε-amino-caproyl-NH₂-Separon, and the constitutively-produced β-glucanase system and some properties of purified β-1,3-glucanase were compared with those of freely suspended cells. It was found that the mycelial immobilization modulates the composition of the β-glucanase system as well as stimulates the export of a functionally different β-1,3-glucanase.     

A concise synthesis of single‐enantiomer β‐lactams and β‐amino acids using Rhodococcus globerulus

BACKGROUND: Pharmaceutical companies continue to evaluate β‐amino acids and β‐lactams in a range of drug candidates. The development of a highly efficient and selective bioresolution of cyclic β‐lactam substrates could yield enantiopure lactams and β‐amino acids with medicinal potential. The aim of this work was to discover and develop a biocatalyst capable of selectively hydrolysing β‐lactam substrates. RESULTS: Screening of our in‐house culture collection led to the discovery of a microorganism, Rhodococcus globerulus (NCIMB 41042) with β‐lactamase activity. Whole‐cell bioresolutions of the β‐lactams 1–4 were successfully carried out and in all cases enantiomeric excesses of the residual lactam and amino acid product were found to be greater than 98%. For one example, the bioresolution was optimised to operate at 60 g L^?1 substrate concentration with a 20% wt/wt cell paste loading. CONCLUSION: A microorganism, Rhodococcus globerulus (NCIMB 41042), capable of selectively hydrolysing a range of cyclic β‐lactams, has been discovered. A scalable whole‐cell bioresolution process has been developed, leading to the synthesis of multigram quantities of enantiomerically pure β‐lactams and β‐amino acids. Copyright © 2007 Society of Chemical Industry     

Cationic copolymers of α,β‐poly‐(N‐2‐hydroxyethyl)‐DL‐aspartamide (PHEA) and α,β‐polyasparthylhydrazide (PAHy): synthesis and characterization

In the present study the derivatization of two water‐soluble synthetic polymers, α,β‐poly(N‐2‐hydroxyethyl)‐DL ‐aspartamide (PHEA) and α,β‐polyasparthylhydrazide (PAHy), with glycidyltrimethylammonium chloride (GTA) is described. This reaction permits the introduction of positive charges in the macromolecular chains of PHEA and PAHy in order to make easier the electrostatic interaction with DNA. Different parameters affect the reaction of derivatization, such as GTA concentration and reaction time. PHEA reacts partially and slowly with GTA; on the contrary the reaction of PAHy with GTA is more rapid and extensive. The derivatization of PHEA and PAHy with GTA is a convenient method to introduce positive groups in their chains and it permits the preparation of interpolyelectrolyte complexes with DNA. © 2000 Society of Chemical Industry     

11α hydroxylation of 16α, 17‐epoxyprogesterone in biphasic ionic liquid/water system by Aspergillus ochraceus

BACKGROUND: The improved efficiency of steroid biotransformation using the biphasic system is generally attributed to the positive effect on the solubility of substrate in aqueous media. A promising alternative for the application of organic solvents in biphasic systems is the use of ionic liquids (ILs). This study aims to investigate the applicability of the biphasic ILs/water system for 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by Aspergillus ochraceus. RESULTS: Of the seven ILs tested, [C₃mim][PF₆] exhibited the best biocompatibility, with markedly improved biotransformation efficiency. In the [C₃mim][PF₆]‐based biphasic system, substrate conversion reached 90% under the condition in which buffer pH, volume ratio of buffer to ILs, cell concentration, and substrate concentration were 4.8, 10/1, 165 g L^?1 and 20 g L^?1, respectively. This is more efficient than that of the monophasic aqueous system. The effects of the cations and anions of these ILs on the 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by A. ochraceus is also discussed. CONCLUSION: The above results showed that IL/water biphasic system improved the efficiency of 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by A. ochraceus, thus suggesting the potential industrial application of ILs‐based biphasic systems for steroid biotransformation. © 2012 Society of Chemical Industry     

Enantiopure N‐Benzyloxycarbonyl‐β2‐amino Acid Allyl Esters from Racemic β‐Lactams by Dynamic Kinetic Resolution using Candida antarctica Lipase B

The dynamic kinetic resolution of α‐substituted racemic β‐lactams by alcoholytic ring‐opening, catalyzed by immobilized lipase B from Candida antarctica is described. With this process, a variety of racemic α‐substituted N‐Cbz‐azetidinones (Cbz=benzyloxycarbonyl) was transformed to the corresponding N‐Cbz‐protected β²‐amino acid allyl esters with high enantioselectivity (up to 99%) and high yields (up to quantitative) at room temperature.

     

Negative effect of stretching on the development of β‐phase in β‐nucleated isotactic polypropylene

On the premise that shear in the slit die of an extruder was minimized as far as possible, β‐nucleated isotactic polypropylene (iPP) was extruded. Simultaneously, once the extrudate (in the melt state) left the die exit, it was stretched at various stretching rates (SRs). For iPP with a low content of β‐nucleating agent (β‐NA), the crystallinity of β‐phase (X_β) initially increases with increasing SR, and then decreases slightly with further increase in SR. However, for iPP containing a higher content of β‐NA, with increasing SR, X_β decreases monotonically, indicating a negative effect of SR on β‐phase formation. Small‐angle X‐ray scattering and polarized optical microscopy experiments reveal that, when SR is less than 30 cm min^?1, the increasing amount of row nuclei induced by increasing SR is mainly responsible for the increase of X_β. In contrast, when SR exceeds 30 cm min^?1, the overgrowth of shish structures unexpectedly restrains the development of β‐phase, and spatial confinement is considered as a better explanation for the suppression of β‐phase. Copyright © 2011 Society of Chemical Industry     

Structural characterization of α‐ and β‐nucleated isotactic polypropylene

Modification of isotactic polypropylene (iPP) with two nucleation agents, namely 1,3:24‐bis(3,4‐dimethylobenzylideno) sorbitol (DMDBS) (α‐nucleator) and N, N′‐dicyclohexylo‐2,6‐naphthaleno dicarboxy amide (NJ) (β‐nucleator), leads to significant changes of the structure, morphology and properties. Both nucleating agents cause an increase in the crystallization temperature. The efficiency determined in a self‐nucleation test is 73.4 % for DMDBS and 55.9 % for NJ. The modification with NJ induces the creation of the hexagonal β‐form of iPP. The addition of DMDBS lowers the haze of iPP while the presence of NJ increases the haze. Copyright © 2004 Society of Chemical Industry     

Organocatalytic Enantioselective Michael Reaction of Malononitrile with β,β‐Disubstituted Nitroalkenes

We have developed and optimized an enantioselective Michael reaction of malononitrile with β,β‐disubstituted nitroalkenes. This reaction was catalyzed by a cinchona alkaloid derived thiourea catalyst, producing products of high yields (up to 98 %) and stereoselectivities (up to 93 % ee). One of the adducts was used as an intermediate for the synthesis of dihydropyrrole derivative bearing a synthetically valuable quaternary chiral center.

     

Regio‐ and Enantioselective Copper‐Catalyzed 1,4‐Conjugate Addition of Trimethylaluminium to Linear α,β,γ,δ‐Unsaturated Alkyl Ketones

A regio‐ and enantioselective copper‐catalyzed 1,4‐conjugate addition of trimethylaluminium to linear δ‐aryl‐substituted α,β,γ,δ‐unsaturated alkyl ketones was developed. A series of γ,δ‐unsaturated alkyl ketones were obtained in good yields with high regio‐ and enantioselectivity (up to 88% ee and 96:4 dr). Expansion of the reaction scope to substrates containing aromatic heterocycles also afforded good yields and enantioselectivities (up to 91% ee) with very high regioselectivities, exclusively providing the single 1,4‐products.

     

Highly Enantioselective Michael Addition of Cyclic 1,3‐Dicarbonyl Compounds to β,γ‐Unsaturated α‐Keto Esters

A highly enantioselective Michael addition of cyclic 1,3‐dicarbonyl compounds to β,γ‐unsaturated α‐keto esters catalyzed by amino acid‐derived thiourea‐tertiary‐amine catalysts is presented. Using 5 mol% of a novel tyrosine‐derived thiourea catalyst, a series of chiral coumarin derivatives were obtained in excellent yields (up to 99%) and with up to 96% ee under very mild conditions within a short reaction time.     

Chemoenzymatic Preparation of Enantiopure Homoadamantyl β‐Amino Acid and β‐Lactam Derivatives

Racemic cis‐10‐azatetracyclo[7.2.0.1^2,6.1^4,8]tridecan‐11‐one was prepared from homoadamant‐4‐ene by chlorosulfonyl isocyanate addition. The transformation of the β‐lactam to the corresponding β‐amino ester followed by Candida antarctica lipase A‐catalyzed enantioselective (E>>200) N‐acylation with 2,2,2‐trifluoroethyl butanoate afforded methyl (1R,4R,5S,8S)‐5‐aminotricyclo[4.3.1.1^3,8]undecane‐4‐carboxylate and the (1S,4S,5R,8R)‐butanamide with>99% ee at 50% conversion. Alternatively, transformation of the β‐lactam to the corresponding N‐hydroxymethyl‐β‐lactam and the following Pseudomonas cepacia (currently Burkholderia cepacia) lipase‐catalyzed enantioseletive O‐acylation provided the (1S,4S,6R,9R)‐alcohol (ee=87%) and the corresponding (1R,4R,6S,9S)‐butanoate (ee>99%). In the latter method, competition for the enzyme between the (1R,4R,6S,9S)‐butanoate, 2,2,2‐trifluoroethyl butanoate and the hydrolysis product, butanoic acid, tended to stop the reaction at about 45% conversion and finally gave racemization in the (1S,4S,6R,9R)‐alcohol with time.     

Organocatalytic Enantioselective Aziridination of α‐Substituted α,β‐Unsaturated Aldehydes: Asymmetric Synthesis of Terminal Aziridines

The first example of a highly enantioselective organocatalytic aziridination of α‐substituted α,β‐unsaturated aldehydes is presented. The reaction is catalyzed by simple chiral amines and gives access to highly functional terminal azirdines containing an α‐tertiary amine stereocenter in high yields and enantiomeric ratios (95.5:4.5–98:2).     

Synthesis and Pharmacological Evaluation of α4β2 Nicotinic Ligands with a 3‐Fluoropyrrolidine Nucleus

Nicotinic acetylcholine receptors (nAChRs) play an important role in many central nervous system disorders such as Alzheimer’s and Parkinson’s diseases, schizophrenia, and mood disorders. The α₄β₂ subtype has emerged as an important target for the early diagnosis and amelioration of Alzheimer’s disease symptoms. Herein we report a new class of α₄β₂ receptor ligands characterized by a basic pyrrolidine nucleus, the basicity of which was properly decreased through the insertion of a fluorine atom at the 3‐position, and a pyridine ring carrying at the 3‐position substituents known to positively affect affinity and selectivity toward the α₄β₂ subtype. Derivatives 3‐(((2S,4R)‐4‐fluoropyrrolidin‐2‐yl)methoxy)‐5‐(phenylethynyl)pyridine ( 11 ) and 3‐((4‐fluorophenyl)ethynyl)‐5‐(((2S,4R)‐4‐fluoropyrrolidin‐2‐yl)methoxy)pyridine ( 12 ) were found to be the most promising ligands identified in this study, showing good affinity and selectivity for the α₄β₂ subtype and physicochemical properties predictive of a relevant central nervous system penetration.