Cloning and Expression of clt Genes Encoding Milk-Clotting Proteases from Myxococcus xanthus 422

Abstract

The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.Currently, there are three main types of rennins used by the cheese-making industry: (i) rennins extracted from the abomasum of suckling ruminants (such as chymosin [EC 3.4.23.4]), (ii) rennins prepared from microbial broths, and (iii) recombinant rennins. In general, animal rennins are not sufficient to cover world demands, and this fact has prompted research into both microbial and recombinant rennins. Although a variety of bacteria, yeasts, and fungi have been isolated as natural producers of milk-coagulating enzymes (3,10,13,26,32,45), only two genera are used worldwide in cheese production: Mucor (12, 39) and Endothia (8, 15). Several aspartic protease genes from molds and bacteria have been cloned and expressed (15,22,33,44). Bovine chymosin has been cloned and successfully expressed in

Combined milk gel generated with a novel coagulating enzyme by Virgibacillus sp. SK37, a moderately halophilic bacterium

“…), Myxococcus xanthus 422 (Poza et al . ) and others. The chestnut blight fungus ( Cryphonectria parasitica ) cleaves the Ser 104 ‐Phe 105 bond (Jacob et al .…”

“…Microbial milk-clotting enzymes are an alternative to expensive calf rennet and its recombinant counterparts. Sources of fungal enzymes include Penicillium citrinum (Abdel-Fattah et al 1972), Mucor pusillus (Nouani et al 2009), Myxococcus xanthus 422 (Poza et al 2004) and others. The chestnut blight fungus (Cryphonectria parasitica) cleaves the Ser 104 -Phe 105 bond (Jacob et al 2011), while the Rhizomucor protease hydrolysed mostly the b-casein (Broome et al 2006).…”

Optimization of conditions for expression and activation of a splice variant of prochymosin lacking exon 6 in Escherichia coli

“…At present, calf chymosin is the only recombinant milk coagulant used in cheese production Neelakantan et al 1999;Poza et al 2004). According to biochemical and genetic evidences the recombinant chymosins are identical to calf rennet.…”

Proteins Associated with theMyxococcus xanthusExtracellular Matrix

“…MXAN3885 is spore coat protein U, a protein known to be assembled on spores (24). MXAN2791 is a zinc metalloprotease known as protease B that has been identified in two studies examining milk-clotting ability by E. coli containing M. xanthus genomic DNA libraries (41,42). A C-terminal portion of MXAN0075 and the entire length of MXAN2375 share homology to the amidohydrolase family, subgroup 1 (pfam01979).…”