Safety evaluation of a novel muramidase for feed application.

Safety evaluation of a muramidase produced by a Trichoderma reesei strain (safe lineage), expressing a muramidase gene isolated from Acremonium alcalophilum is presented. Intended use in feed of this enzyme is as digestive aid in broiler chickens. Muramidase 007, was non-mutagenic and non-clastogenic in vitro, and no adverse effects were observed in 90-day subchronic toxicity studies in rats at doses up to 1132 mg TOS/kg body weight/day. The enzyme did not exhibit, in vitro, skin, nor eye irritation potential. Acute aquatic toxicity evaluated on daphnia and algae showed absence of effect of the enzyme at the standard doses tested. Muramidase 007 was fully tolerated by broiler chickens in a 6-weeks tolerance study showing no adverse effects in any of the dietary treatments (0, 1×, 5× and 10× maximum recommended dose). In conclusion, Muramidase 007 is found to be toxicologically inert, and there are no worker's safety concerns if standard precautions are instituted and a non-dusty formulation is employed. Muramidase 007 is well tolerated by the target species (broiler chickens) and cause no harm to the environment. The beneficial safety evaluation of Muramidase 007 is in line with this type of enzyme that is found ubiquitously in nature.

Exchanging the active site between phytases for altering the functional properties of the enzyme.

By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase-1, that was 15-26 degrees C more thermostable than the parent fungal phytases used in its design (Lehmann et al., 2000). We now sought to use the backbone of consensus phytase-1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site (i.e., all the divergent residues) with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH-activity profile. For the new protein termed consensus phytase-7, a major - although not complete - shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach. Although the exchange of the active site was associated with a 7.6 degrees C decrease in unfolding temperature (Tm) as measured by differential scanning calorimetry, consensus phytase-7 still was >7 degrees C more thermostable than all wild-type ascomycete phytases known to date. Thus, combination of the consensus approach with the selection of a "preferred" active site allows the design of a thermostabilized variant of an enzyme family of interest that (most closely) matches the most favorable catalytic properties found among its family members.

An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha.

An efficient process was developed for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups, previously reported as repressive substrates, were used as main carbon sources during fermentation. Glucose was even the most productive substrate for high-level production of phytases. Compared with the process using glycerol, the standard carbon source used for this process until now, the use of glucose led to a reduction of more than 80% in the raw materials costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g/L) were obtained in the medium, with phytase representing over 97% of the total accumulated protein. These levels greatly exceed those reported so far for any yeast-based expression system. Very efficient downstream processing procedures were developed with product recovery yields over 90%. Both the fermentation and downstream processing were successfully tested in pilot scale up to 2000 L. As a result, H. polymorpha can be used as a highly competitive system for low-cost phytase production.