Detergent enzymes optimization

Many of the above enzymes require detergents for optimal activity in vitro. Some workers have suggested that naturally occurring substances may be present in mammalian cells which could serve a similar function. It has also been postulated that the various sphingolipid hydrolases may also be aligned in an ordered fashion on lysosomal membranes. In that case hydrolysis of sphingolipids should be much more efficient than random access of various substrates to enzymes freely admixed within a lysosomal sac (Brady, 1978). 

All of the preceding techniques are extensively used for the production of detergent enzymes such that today the vast majority of industrial enzymes are produced by recombinant techniques. This is done in a limited number of optimized, well-known production hosts. Some of the most frequently used host organisms are the Bacillus species—B. subtilis, B. licheniformis, and B. clausii, which are used for production of proteases and amylases. The lipases and cellulases of fungal origin are produced by cultivation of the filamentous fungi Aspergillus oryzae and Trichoderma reesei. 

Contrary to the commodity chemical business, the key to win in the specialty products market does not lie in squeezing out profits by means of economies of scale or process optimization. Rather, it lies in the ability for fast new product launches in order to capture the largest market share as soon as possible. Since superior product quality and performance is what really differentiates one specialty product from another, the product properties need to be adjusted as required by business needs. For example, the ability to manipulate functional chemicals in detergent products such as enzymes and zeolites, as well as backbone chemicals like surfactants, is often the key to success for both the detergent manufacturers and chemical suppliers [3], This trend has created an urgent need for an efficient and effective product and process development for these products. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

The enzyme, which was extractable from activated but not from resting PMNs, exhibited a quadratic dependence on enzyme concentration which could be restored to a linear relationship by the addition of phosphatidyl ethanolamine. This phospholipid may have been removed by treatment with detergent. The K of the enzyme in Triton X-lOO in the presence of FAD was greater for NADH (930 pM) than for NADPH (33 pM) and the optimal pH was broad with a maximum at 7.0 Its activity in the presence of phosphatidyl ethanolamine was judged sufficient to account for all of the O which activated PMNs produced. 

Phosphotransferase activity of the enzyme has been found to respond much more extensively than does phosphohydrolase activity to detergent treatment (see, for example, references 86, 89, 92). This observation led us to investigate in detail alterations in catalytic properties of both phosphotransferase and phosphohydrolase activities produced by detergent treatment. Deoxycholate supplementation has been found to shift the pH optimum of PPi-glucose phosphotransferase from approximately 4.5 noted with fresh microsomal suspensions to approximately pH 5.7 with optimal (0.20%, w/v) deoxycholate concentrations added to micro-somes (see Fig. 2 and references 80 and 98). Preparations obtained by... 

In biochemical assays, additives such as detergents, DMSO, urea, BSA, and glycerol are commonly used to improve reaction performances and enzyme stability. However, these additives also act as crystallization disturbing agents preventing the formation of optimal crystals for the MALDI process. Analytical sensitivity and mass accuracy can be affected. The challenge is to develop bioassays that can perform optimally without crystallization disturbing additives. Often, it is necessary to use elaborate purification processes prior to analysis. 

The definition of a more efficient enzymatic system could be based on the separation of the catalytic cycle of the enzyme and the degradation step by the Mn3+ reactive species in MnP systems. The Mn3+-chelates present several advantages in their use as oxidants. They are more tolerant to protein denaturing conditions such as extremes of temperature, pH, oxidants, organic solvents, detergents, and proteases, and they are smaller than proteins therefore, they can penetrate microporous barriers inaccessible to proteins. The optimization of the production of the Mn3+-chelate will have to be compatible with the minimal consumption and deactivation of the enzyme. 

Figure 3. Frequency dependence of electric-field-stimulated ATP hydrolysis activity of a highly purified Ecto nucleotide diphosphohydrolase. The ATP splitting activity of the detergent-solubilized Ecto enzyme was measured at 37 °C in the absence of acfield (a,), in the presence of 5.0-V/cm (peak-to-peak) ac field (O), and in the presence of 10-mM vanadate (a). Buffer 0.1% of detergent NP40, 30-mM histidine, 4-mM ATP, 4-rnM Mg2+, at pH 7.4. The optimal amplitude of stimulation is 5.0 V/cm with a 10-kHz ac field. (Adapted from...

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