Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris

Background: Human is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (MutS) and X33 (Mut+) strains of Pichia pastoris P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression. Results: In the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions. Conclusions: This study demonstrated that MutS strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut+ strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris.

Viability of probiotic bacteria and some chemical and sensory characteristics in cornelian cherry juice during cold storage

Background: Increased popularity of vegetarianism, lactose intolerance, and the high cholesterol content in dairy products, are all factors that have recently increased the demand for nondairy probiotic products. The objective of this study is to evaluate the effect of refrigeration on the viability of probiotics and asses someof the chemical and sensory characteristics in cornelian cherry juice. Results: The Iranian native probiotic strain (L. casei T4) showed greater viability compared to industrial types (viable count of 8.67 log cfu/mL versus <6.0 log cfu/mL at d 28). However, this most tolerant Iranian strain, could not withstand the conditions of ‘Natural juice’ at pH 2.6 for more than 7 d. Following a pH adjusted treatment (to pH ~3.5), the viability of the strain was improved to 28 d with some evidence of increased growth of the probiotic. However, the level of antioxidant activity, anthocyanin and phenolic compounds, revealed a slight decrease during cold storage. The changes in the chemical profile of the sample containing L. casei T4 indicated fermentation activity during cold storage. Sensory evaluation results showed significant differences between samples containing L. casei TD4 and other samples in taste, odor and overall acceptance in a complimentary way. Conclusions: The results showed that low pH and presence of inhibitor phenolic compounds of cornelian cherry juice have negative effect on viability of probiotics, especially industrial strains during refrigerated storage.