Mycelium-bound lipase production of immobilized whole cell from a wild-type Penicilium citrinum strain
Abstract
Pencillium citrinum whole cell was cultivated and immobilized in a low cost support aiming to be used as an active and stable biocatalyst for modification of oil and fats. PUF (polyurethane foam) coated in 6mm was used as support and the immobilization occurred as a natural consequence of cell growth. Olive oil was the mycelium-bound lipase inductor and the production was optimized by statistical analysis of pH and temperature effects in culture broth. Immobilized whole cell was characterized as a natural immobilized lipase and all the assays were made using olive oil hydrolysis. The mycelium-bound lipase production was improved by adjustment the culture broth to pH 7.5 and 35°C of incubation temperature. The SEM micrographs showed the entangled cells morphology and a high adhesion in support matrix. Biochemical characterization revealed maximum values of lipase activity in pH 8 and 40°C and a half-life time at 60°C was 2.2h. Results from kinetics study indicated the biocatalyst follow the Michaelis-Menten kinetic. The potential catalytic of immobilized whole cells was assessed in soybean oil hydrolysis and 55.7% of degree of hydrolysis was attained in 12h.
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Introduction
Generally, enzymes associated with the mycelia are referred to as mycelium-bound enzymes. Mycelium-bound enzymes are economically attractive because they can be produced at low cost and considered as naturally immobilized enzymes that may be used without the laborious operations of isolation, purification and addition of co-factors, etc [1]. The whole-cell biocatalyst includes the cell wall- or the cell membrane-bound lipase (i.e., intracellular enzyme), which can be used instead of extracellular lipase [2]. Over the past decades, mycelium-bound lipase of filamentous fungus has been extensively studied as an alternative biocatalyst in biotransformation processes [3].
Mycelium-bound lipase can be used directly as suspended free cells or immobilized within biomass support particles as whole cells biocatalyst [4]. Immobilization of whole cells provides stability as well as improves reusability [5]. The concept of immobilized whole cells is different for those applied in fermentation process, since whole cells keep the enzymes in active state, without cell replication. Whole cell immobilization requires the same techniques applied to conventional enzyme immobilization, such as adsorption, covalent attachment, cross-link and entrapment, although the most useful for filamentous fungi is the entrapment in porous support, due to its morphology [3].
The literature has shown that whole cells immobilization in reticulated polyurethane foam (PUF) is a convenient way to spontaneously obtain immobilized whole cells. So far, a few strains were studied as potential mycelium-bound lipase producers, such as Rhizopus sp. [6, 7, 8], Aspergillus sp. [9, 10, 11] and Mucor sp. [12, 13, 14]. The genus Penicillium is also a known fungal producer of lipases which are employed in the dairy industry and in a number of bioconversions of industrial importance [15], nevertheless its potential as whole cells biocatalyst were not enough explored.
In this context the present work aims to investigate the potential of a wild-type Penicillium citrinum strain as a myceliumbound lipase producer and its immobilization in PUF. The objective was to evaluate the catalytic potential of the immobilized whole cells in enzymatic modification of fats and oils, such as hydrolysis, transesterification and interesterification processes. For this, firstly, the physicochemical parameters were optimized to enhance the production of mycelium-bound lipase and then the immobilized whole cells produced were characterized as a conventional immobilized lipase. Additionally, the efficiency biocatalytic process of soybean hydrolysis with immobilized whole cells was evaluated on batch reaction.
Conclusion
P. citrinum whole cells were successfully immobilized in PUF and suitable conditions to enhance the mycelium-bound lipase production were determined. Maximum lipase activity was achieved in 96 h incubation time in medium at pH=7.5 at 30 °C using inoculum size of 30 mL L -1 . SEM images showed strong adhesion of cells into the support matrix under the establishment conditions. The immobilized whole cells were characterized and revealed optimum lipase performance in pH 8.0 at 37 °C, with Vmax 123.24 U g-1 and satisfactory thermal stability at 60° C (half-life time =2.23 h). Enzymatic soybean hydrolysis was carried out by immobilized whole cells, which obtained maximum hydrolysis degree of 55.7± 1.56% in 12h of reaction time. These results suggest the use of immobilized whole cells as biocatalyst are promising and economically attractive in oils and fats industry.