Bioprocessing and immobilization of cell envelope proteinases from Lactobacillus delbrueckii subsp. lactis 313, for protein degradation

Proteolytic enzymes are a useful class of biomolecules due to their ubiquity and the plethora of physiological roles they play in living systems. These enzymes are esponsible for the breakdown of proteins to peptides and have several applications in food, pharmaceuticals, diagnostics, photographic, waste treatments, bioremediation, and in the textile industry. Cell-envelope proteinases (CEPs) are a special class of industrially relevant extracellular proteolytic enzymes obtained from lactic acid bacteria. In the food industry, CEPs have been known to improve the texture and organoleptic characteristics of dairy products and also have the potential to release bioactive peptides encrypted in dairy proteins. However, research is lacking on detailed optimisation of fermentation parameters essential for the generation of CEPs of organisms in the genera Lactobacillus. The proteolytic system and CEPs from lactobacilli are also not fully characterized and further; the use of free CEPs in industrial processes is currently suboptimal and presents certain drawbacks such as poor operational stability. Regulatory requirements such as that of the United Nations Food and Agriculture Organization demand the separation of enzymes from certain food and pharmaceutical products when enzymes are involved in the production process. This is difficult to do, if not impossible, for soluble enzymes. These make soluble CEP-based process economically unfeasible, especially when combined with the usual challenges associated with the use of most soluble enzymes, i.e. high cost, poor stability, and lack of multiple utility. This study therefore explored the production of CEPs expressed in Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) in a cost effective manner. Immobilization techniques were also deployed for the design and production of cheap, reusable and stable biocatalysts of LDL 313 CEPs, for use in protein degradation. LDL 313 was an understudied bacterium, thus, initial work considered it’s growth characterization for the purpose of CEP production. As expected, cell growth was dependent on fermentation conditions such as temperature and initial pH. However, cell growth rates under anaerobic conditions were markedly higher than growth under microaerophilic conditions. The caseinolytic specificity of LDL 313 CEPs was also identified. Being proteolytic for for β -casein and κ-casein, CEPs from LDL 313 were classified as class I CEP (CEPI)of the lactococcal proteinase classification system. Studies were also done to optimize the batch culture conditions that enhance CEPs expression. Using a combination of conventional sequential techniques, the batch growth conditions (inoculum concentration, culture agitation speed, incubation temperature, starting pH, and carbon/nitrogen ratio of production medium) were optimized, for the first time, to ensure profuse CEP production in LDL 313. Moreover, since CEPs are cell-envelope-bound enzymes they are relatively easy to extract from lactic acid bacteria cells. When CEP extraction was studied with different extraction agents, 5 M LiCl was observed to be the most suitable. Sub-cellular localization studies also showed that about 95% of CEP activity was detected in cell-wall fractions implying that CEPs in LDL 313 are located in the peptidoglycan cell-wall. Together, these results provide insights into conditions and parameters that ensure optimum cell growth, high CEP yields and extraction protocol to release high levels of CEPs. Following this, two different strategies for making stable biocatalysts from CEPs were explored, namely, the immobilization of enzymes onto a fabric carrier, and, cross-linking enzyme aggregates. Firstly, CEPs and trypsin (as a model enzyme in comparison with CEP) were immobilized in a simple, cheap and quick approach onto polyester via support functionalization with ethylene diamine and cross-linking with glutaraldehyde. Secondly, cross-linked enzyme aggregates were prepared from CEPs via coupled precipitation/cross-linking with ammonium sulphate and glutaraldehyde respectively. The immobilized biocatalysts had good activity characteristics and properties. For example, immobilized enzymes had good recovered activity (85% for CEP immobilized on polyester; and ~ 22% and ~ 41% respectively for CLCEPA prepared in the absence and presence of proteic feeders). They also had the ability to be used at other than usual conditions such as high temperatures (40 - 70 °C); and organic solvent conditions (CLCEPA still retained activity in 20 – 100 % ethanol/buffer mixtures). Both immobilized enzymes had the ability to be recycled (CLCEPA retained ~ 22% initial activity whereas CEPs immobilized on polyester retained ~ 41% when recycled 5 times. Additionally, both immobilized biocatalyst had proteolytic properties, effectively hydrolysing several macromolecular proteins substrates (casein, bovine serum albumin, whey protein isolate, β-lactoglobulin, skimmed-milk protein and chicken egg albumin). The immobilized CEPs biocatalysts could be utilized for protein degradation to obtain two streams of industrially relevant products namely protein/peptide-rich product mixture from casein; and whey protein/peptide-based surface active foams – both of which can be used for food applications. In summary, the potential of Lactobacillus delbrueckii subsp. lactis 313 to produce cell-envelope proteinases was explored and the development of stable forms of these enzymes was studied, optimized and tested with several food proteins. The outcome is a whole systems approach to developing and establishing an enzyme framework for protein degradation in a cheap cost-effective manner. This therefore has the potential for various industrial applications in protein degradation and/or peptide production.