Erden-Karaoglan, Fidan

Pichia pastoris is among the most popular expression systems for recombinant protein production. The ability to secrete high titers of recombinant proteins is one of its primary advantages. Co-expression of folding-assisting factors is considered one of the strategies to improve protein production with this expression system. In this study, the effect of PDI, KAR2, HAC1, and ERO1 co-expression on Geobacillus stearothermophilus lipase production in P. pastoris was investigated. The chaperones were co-expressed under the regulation of the GAP promoter in clones with different lipase production levels (LPS#2 and LPS#8). Results showed that lipase-PDI co-expression clones have the highest activity. The effect of the other chaperones had varying effects for LPS#2 and LPS#8 clones. LPS#2-PDI and LPS#8-PDI were further analyzed to determine the effect of dimethyl sulphoxide (DMSO). Different concentrations (0.5%, 1%, and 2%) of DMSO were tested. The highest activity was obtained with approximately 1.5-fold activity in the LPS#8-PDI clone with 0.5% and 1% DMSO concentration levels. Comparison of the fermentation kinetic parameters revealed that the activity level of 56.54 U/mL provided with LPS#8 increased to 73.84 U/mL in the LPS#8-PDI clone with PDI co-expression, and when PDI co-expression was combined with DMSO conditions (0.5%), it reached 114.46 U/mL. It was observed that the productivity (U/g wet cell/g substrate/h) of the LPS#8 clone was increased 1.27-fold with LPS#8-PDI and 2.05-fold with LPS#8-PDI-DMSO conditions. In future studies, it is possible to achieve higher protein production levels by optimization at the fermenter scale.

Çalışkan, Büşra; Demir, Yeliz; Türkeş, Cüneyt

Glaucoma is a neuropathy disorder and is generally treated by drugs. Allergic conjunctivitis is a common ophthalmologic disease. Paraoxonase 1 (PON1) is an organophosphate hydrolyzer and antiatherogenic enzyme. PON1 is known for preventing atherosclerosis through lipid-modifying features, as well as which has decisive actions of antiapoptosis, anti-inflammatory, antithrombosis, and antiadhesion antioxidant activity properties. Thus, reducing the enzyme levels in hyperthyroidism, chronic renal failure, glaucoma, diabetes mellitus, and cardiovascular diseases is a significant risk. This study was tested some ophthalmic drugs used to treat the diseases, such as glaucoma and allergic conjunctivitis, mentioned above, travoprost, latanoprost, ketotifen, emedastine, and olopatadine, for their inhibition activities against PON1. These drugs displayed the potent inhibition effect with IC50 values ranging between 14.95 ± 0.15 and 299.60 ± 4.07 μM and KI constants ranging from 9.71 ± 2.63 to 261.50 ± 59.98 μM. Besides, the molecular docking analyses of the competitive inhibitors, travoprost, emedastine, and olopatadine, were performed to understand the binding interactions on the enzyme's binding site. According to both in vitro and in silico analysis results, travoprost had the most potent effect on PON1 enzyme activity.

Demir, Yeliz; Durmaz, Lokman; Taslimi, Parham; Gulçin, İlhami

Aldose reductase (AR), α-amylase, and α-glycosidase are vital enzymes to prevent diabetic complications. Here, AR was purified from sheep kidney using elementary methods with 111.11-purification fold and with 0.85% purification yield. The interactions between some phenolic compounds and the AR, α-glycosidase, and α-amylase enzyme were determined. It was found that phenolic compounds exhibit potential inhibitor properties for these enzymes. For α-amylase, studied phenolic compounds showed IC50 values in the range of 601.56–2,067.78 nM. For α-glycosidase, Ki values were found in the range of 169.25 ± 27.22–572.88 ± 106.76 nM. For AR, Ki values in the range of 8.48 ± 0.56–43.26 ± 7.63 µM. However, genistein showed the best inhibition effect toward AR and α-glycosidase, but delphinidin chloride exhibited the best inhibition effect against α-amylase enzyme. We determined that all compounds showed noncompetitive inhibition effect against AR and α-glycosidase. Also, studied phenolic compounds may be useful in the prevention or treatment of diabetic complications.