Recombinant expression of Clostridium sporogenes autolytic enzymes in Escherichia coli

One of the most significant fermentation defects, late blowing is caused on by bacteria of the genus Clostridium and is most frequently seen in hard and semi-hard cheeses with high pH levels. It can cause economic losses for the cheese industry. The production of gases like carbon dioxide and hydrogen as the cheese ripens generally causes the cheese matrix to be unable to withstand the pressure of the gas, leading to pores, cracks, and crevices in the cheese's structure. Along with these gases, certain metabolites like butyric acid and acetic acid might also result in an unfavorable, atypical cheese flavor. Kaşar cheese production in our nation's dairy industry has encountered a significant number of late blowing concerns in recent years. Occasionally, this scenario can be directly reflected in consumer complaints that cheddar cheese swells before it expires. Although techniques like bactofugation, which reduces bacterial spores in raw milk, the use of lysozyme and nisin as additives, and the use of protective starter cultures have been used to develop solutions for this issue, it is difficult to say that the problem has been fully resolved. Various alternatives to these techniques should therefore be developed. By analyzing the lytic effect of the autolytic enzymes, which are site-specific and particularly effective in Gram-positive bacteria, on the inhibition of Clostridium strains, one of the most significant deterioration factors in cheese production, the goal of this thesis study is to contribute to the solution of the late blowing problem in cheese. This thesis describes the first cloning and expression of two autolytic enzymes from the genome of Clostridium sporogenes cells isolated from local Kaşar cheese samples: spore cortex lytic enzyme (SleB) and D-alanyl-D-alanine Carboxypeptidase (CRB).The study revealed a partial inhibition of recombinant autolytic enzymes on C. sporogenes strains, and it was concluded that additional research might increase the activity of these enzymes and potentially replace the late blowing issue in cheese production