Protease production from marine microorganism by immobilized cells

Proteases represent the most important kind of enzymes from an industrial point of view. Alkaline proteases have applications in leather processing, laundry detergents, production of protein hydrolysates, and food processing. In this study, cell immobilization has been used for enhancing enzyme titer. Our aim was to choose carriers, methods, and optimal conditions for immobilization of growing cells of T. turnirae and evaluate the immobilized biocatalysts in repeated batch fermentations for production of alkaline protease. Teredinobacter turnirae cells were adsorbed onto different matrices namely, ceramic support – CeramTec F1/porous PST 5, broken pumice stone (Particle size: 1.5-2.5 mm, 2.5-3.5 mm, 5-15 mm) and Silicone foam ImmobaSil D. These matrices effectively retained biomass, and increased volumetric productivity by over 207%, when compared to free cells. Enzyme productivity by immobilized cells in ceramic support (CeramTec) and Silicone foam ImmobaSil D, was about 2.1 fold higher than the corresponding free cells. Repeated batch production of alkaline protease by immobilized cells in Silicone foam ImmobaSil D matrix was achieved for 5 repeated batches without a significant decrease in the production of protease enzyme. Moreover, electron micrograph images indicate how T. turnirae colonizes porous support matrices. Introduction Proteases are a complex group of enzymes collectively know as peptidyl peptide hydrolases (EC 3.4.21.14) and constitute one of the most important groups of industrial hydrolytic enzymes. This enzyme occupies about 60% of the global enzyme market (Sachidanandham et al., 1999). These may be extracellular or intracellular in nature and degrade proteins to peptides and amino acids. These enzymes posses a wide variety of physiochemical and catalytic properties. Alkaline proteases have applications in leather processing, laundry detergents, production of protein hydrolysates, and food processing (Godfrey and Reichet 1985; Zukowski 1992). A major trend in the detergent industry is a shift to nonphosphate detergent formulations. Because of concerns about their ecological effect the phosphate builders in phosphate detergents have been replaced with builders that tend to decrease the cleaning power of the detergent formulations and increase the pH of their suds to the range 10-12 rather than the typical value of about 9 for phosphate detergents. To replace the lost cleaning power detergent makers have turned to proteases. However, the availability of proteases that posses high activity and stability in the required pH range is limited. The shipworm protease is highly active and stable in this pH range and increases the cleaning power of a standard nonphosphate detergent independent of pH over the range 10-11.9 better than the common commercial protease additive subtilisin. In this study, a novel protease was produced by Teredinobacter turnirae, a new isolate symbiotic bacterium found in the gland of Deshays of the marine shipworm Psiloteredo healdi (Waterbury et al., 1983). The secreted protease is somewhat unusual because it has an alkaline iso-electric point. It exhibits complete thermal stability up to 40 °C, but retains a high level of activity above 50 °C for at least 60 min. The enzyme remains active over a broad pH range and exhibits salt tolerance up to saturated sodium chloride (Griffin et al., 1992). Additionally, the produced protease is resistant to the trypsin inhibtor PMSF, which reacts with active site serine residues. These properties render the produced protease useful in detergents and other low-moderate temperature industrial applications. One of the main problems concerning the batch process of T. turnirae cells is the low yield of protease enzyme. Therefore, the recent research is focused on new approaches for increasing the cell concentration and protease production, respectively. One of the methods applied for maintaining high cell concentration and higher productivities is immobilization (Beshay et al., 2003a; Beshay et al., 2003b). Cell immobilization offers several potential advantages to fermentation systems from the standpoint of process engineering (Navarro and Durand 1977; Helmo et al. 1985; Karel et al., 1985; Brodelius and Vandamme 1987). Like enzyme immobilization, the immobilization of cells, has the same advantages, when comparing the immobilized cells with the free cells. Thus the immobilization process makes possible the reuse or the continuous use of this type of biocatalyst (Zhang et al., 1989; Galazzo and Bailey 1990; Ban et al., 2002). Immobilized cells are often more stable than the equivalent free cells; immobilized cell processes are easier to automate and enable exploitation of the advantages of various reactor configuration. Immobilized cells are convenient to handle, appear to be less susceptible to microbial contamination, and permit easy separation of products from the biocatalyst. The use of immobilized cells enables greater control throughout the reaction. Immobilization also facilitates the use of dense cell populations by altering the rheological properties of the suspending medium. The fluid viscosity is lower than when comparable numbers of cells are freely suspended in solution. Lower viscosities contribute to better mixing and mass transfer properties in the reactor. Our previous work (Beshay & Moreira 2003c) has indicated that porous sintered glass SIRAN is a suitable matrix for the immobilization of T. turnirae cells by means of adsorption. In addition, immobilized cells are capable of producing a high protease activity. Therefore, our aim in this study was to use cheaper immobilization supports and choose suitable carriers, methods and optimal conditions for immobilization of growing cells of Teredinobacter turnirae and to evaluate the immobilized biocatalysts in repeated batch fermentations for production of alkaline protease. Materials and methods Microorganism and cultivation conditions The bacterium Teredinobacter turnirae was generously supplied by Dr. Richard Greene (USDA, Peoria, IL, USA). A basal medium (PlacketBurman media, PB) was used throughout all experiments (Beshay & Moreira 2003b). Growth was carried out with 50-ml cultures in 250-ml Erlenmeyer flasks shaken at 120 rpm and 30 °C. Supporting matrices for cell immobilization Three different inorganic porous supports, ceramic support CeramTec F1/porous PST 5, broken pumice stone and Silicone foam ImmobaSil D were used to immobilize T. turnirae cells. Ceramic support matrix was obtained from CeramTec AG Innovative Ceramic Engineering, Wunsiedel, Germany. It had a diameter of 1.5-2.5 mm, a particle density of 1430 g/l, a pore volume of 0.25 ml/g and a specific surface area of 20 cm/cm. The second carrier (broken pumice stone) was provided by Joseph Raab GmbH & Cie.KG, Neuwied, Germany. Three different particle sizes were used. The chemical composition of broken pumice stone is as follows: Silicic acid SiO2 55%, Aluminium oxide Al2O3 22%, Alkalis K2O+Na2O 12%, Iron oxide Fe2O3 3%, Calcium oxide CaO 2%, Magnesium oxide MgO 1%, Titanium dioxide TiO2 0.5%, Ignition loss 4%. The physical properties of broken pumice stone are summarized in Table 1. Table 1: Characteristics of porous broken pumice stones Carrier Diameter (mm) Pore volume (g/ml) Particle density (g/l) Specific surface area (cm/cm) 1.5 – 2.5 0.93 670 27 Pumice stone 2.5 – 3.5 0.93 670 27