Dispersal Improvement of a Powder Formulation of Penicillium oxalicum, a Biocontrol Agent of Tomato Wilt.

Sugars, polyalcohols, inorganic salts, and detergents were added to conidia of Penicillium oxalicum at three different points of the production-formulation process to improve water dispersal. Effects also were tested on conidial germination and production. Conidial germination without additives ranged from 51 to 79%. Additives did not reduce conidial germination except for 50% polyethylene glycol (PEG) 300 and 10% CaCl2. Sunflower oil and sodium alginate, sucrose (0.5, 15, 30, and 60%), D-sorbitol (30 and 60%), glycerol (2, 5, 20, and 30%), 30% PEG 300, CaCl2 (0.01 to 1%), Tween 20 (0.01, 0.02, 0.5, and 1%), and Tween 80 (0.01 to 1%) enhanced conidial germination. Production without additives ranged from 0.57 to 4.58 conidia × 108 g-1 substrate. Additives did not affect conidial production except for reduction by 60% D-sorbitol, 60% fructose, and 10% CaCl2. Conidial dispersal in water improved when 1.5% sodium alginate was added to substrate in bags before production, and when 1.5% sodium alginate, 60% sucrose, 60% D-sorbitol, 60% fructose, 5 to 20% PEG 8000, or 20% glycerol were added to conidia before drying. Dispersal of dried conidia was enhanced with 1% Tween 20, 1% Tween 80, 1% Trition X-100, 10% Agral, and 1.5% sunflower oil. Two P. oxalicum formulations (conidial suspensions maintained with 60% sucrose or 1.5% sodium alginate for 10 min before drying) significantly reduced tomato wilt caused by Fusarium spp. under greenhouse conditions and, in a preliminary trial, by Verticillium spp. in a field assay.

'Interfacial affinity chromatography' of lipases: separation of different fractions by selective adsorption on supports activated with hydrophobic groups.

Lipases contained in commercial samples of lipase extracts from Rhizopus niveus (RNL) and Candida rugosa (CRL) have been selectively adsorbed on hydrophobic supports at very low ionic strength. Under these conditions, adsorption of other proteins (including some esterases) is almost negligible. More interestingly, these lipases could be separated in several active fractions as a function of a different rate or a different intensity of adsorption on supports activated with different hydrophobic groups (butyl-, phenyl- and octyl-agarose). Thus, although RNL seemed to be a homogeneous sample by SDS-PAGE, it could be separated, via sequential adsorption on the different supports, into three different fractions with very different thermal stability and substrate specificity. For example, one fraction hydrolyzed more rapidly ethyl acetate than ethyl butyrate, while another hydrolyzed the acetate ester 7-fold slower than the butyrate. Similar results were obtained with samples of CRL. Again, we could obtain three different fractions showing very different properties. For example, enantioselectivity for the hydrolysis of (R,S) 2-hydroxy-4-phenylbutanoic acid ethyl ester ranged from 1.2 to 12 for different CRL fractions. It seems that very slight structural differences may promote a quite different interfacial adsorption of lipases on hydrophobic supports as well as a quite different catalytic behavior. In this way, this new 'interfacial affinity chromatography' seems to be very suitable for an easy separation of such slightly different lipase forms.

Immobilization of lipases by selective adsorption on hydrophobic supports.

The preparation of immobilized derivatives of lipases that may be useful to develop industrial processes of organic synthesis is an exciting field of research in which three main features have to be simultaneously considered: (a) immobilized derivatives have to be compatible with very different reaction requirements (e.g. continuous adjustment of pH with concentrated alkali, use of aqueous media or organic solvents, etc.); (b) Sometimes, some activity/stability properties of lipases should be improved during immobilization; and (c) because of a complex mechanism of action, lipases are poorly active in the absence of hydrophobic interfaces. In this paper, we will review different approaches for lipase immobilization mainly related to the further use of immobilized derivatives to carry out enantio and regioselective hydrolysis in high water-activity systems. Special emphasis is paid to the selective adsorption of lipases on tailor-made strongly hydrophobic support surfaces. This new immobilization procedure is based on the assumption that the large hydrophobic area that surrounds the active site of lipases is the one mainly involved in their adsorption on strongly hydrophobic solid surfaces. Thus, lipases recognize these surfaces similarly to those of their natural substrates and they suffer interfacial activation during immobilization. This immobilization method permits: (a) promote a dramatic hyper-activation of most of lipases after their immobilization. That is, adsorbed lipases show very enhanced esterase activity in the absence of additional hydrophobic interfaces; (b) promote highly selective adsorption of lipases, at very low ionic strength, from impure protein extracts. That is, we can associate immobilization and purification of lipases; (c) promote interesting improvements of enantioselectivity after immobilization; and (d) promote a strong but reversible immobilization that enables us to recover these expensive supports after inactivation of immobilized lipases.

A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports

A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus). Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins. At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization. Interestingly, these immobilized lipase molecules show a dramatic hyperactivation. For example, lipases from R. niveus, M. miehei, and H. lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate). Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g. (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester). These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their "open and hyperactivated structure". This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases. Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate). Copyright 1998 John Wiley & Sons, Inc.