MCAD deficiency caused by compound heterozygous pathogenic variants in ACADM

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is an autosomal recessive disease caused by biallelic pathogenic ACADM variants. We report a case of an asymptomatic Japanese girl with MCAD deficiency caused by compound heterozygous pathogenic variants (NM_000016.5:c.1040G > T (p.Gly347Val) and c.449_452delCTGA (p.Thr150ArgfsTer4)). Because the MCAD residual activity in lymphocytes of the patient was below the limit of quantification, both variants are likely to cause complete loss of MCAD enzymatic activity.

Characterization of Cell-Envelope Proteinases from Two Lacticaseibacillus casei Strains Isolated from Parmigiano Reggiano Cheese

In the present work, two cell-envelope proteinases (CEPs) from Lacticaseibacillus casei strains PRA205 and 2006 were characterized at both the biochemical and genetic levels. The genomes of both L. casei strains included two putative CEPs genes prtP2 and prtR1, but only prtR1 was transcribed. The extracted PrtR1 proteinases were serine proteinases with optimal activity at 40 °C and pH 7.5, and were activated by Ca2+ ions. Interestingly, PrtR1 from L. casei PRA205 exhibited high residual activity at pH 4 and at 5 °C, suggesting its possible exploitation for fermented food production. The caseinolytic activity against αS1- and β-casein indicated that both PrtR1s belonged to the PI/PIII type. These PrtR1s cleaved β-casein peptide bonds preferentially when amino acid M or N was present at the P1 subsite and amino acids A and D were at the P1′ subsite. Several bioactive peptides were found to be released from PrtR1 after αs1- and β-casein hydrolysis.

Immobilization of β-Galactosidase by Encapsulation of Enzyme-Conjugated Polymer Nanoparticles Inside Hydrogel Microparticles

Increasing the shelf life of enzymes and making them reusable is a prominent topic in biotechnology. The encapsulation inside hydrogel microparticles (HMPs) can enhance the enzyme’s stability by preserving its native conformation and facilitating continuous biocatalytic processes and enzyme recovery. In this study, we present a method to immobilize β-galactosidase by, first, conjugating the enzyme onto the surface of polymer nanoparticles, and then encapsulating these enzyme-conjugated nanoparticles (ENPs) inside HMPs using microfluidic device paired with UV-LEDs. Polymer nanoparticles act as anchors for enzyme molecules, potentially preventing their leaching through the hydrogel network especially during swelling. The affinity binding (through streptavidin-biotin interaction) was used as an immobilization technique of β-galactosidase on the surface of polymer nanoparticles. The hydrogel microparticles of roughly 400 μm in size (swollen state) containing unbound enzyme and ENPs were produced. The effects of encapsulation and storage in different conditions were evaluated. It was discovered that the encapsulation in acrylamide (AcAm) microparticles caused an almost complete loss of enzymatic activity. Encapsulation in poly(ethylene glycol) (PEG)-diacrylate microparticles, on the other hand, showed a residual activity of 15–25%, presumably due to a protective effect of PEG during polymerization. One of the major factors that affected the enzyme activity was presence of photoinitiator exposed to UV-irradiation. Storage studies were carried out at room temperature, in the fridge and in the freezer throughout 1, 7 and 28 days. The polymer nanoparticles showcased excellent immobilization properties and preserved the activity of the conjugated enzyme at room temperature (115% residual activity after 28 days), while a slight decrease was observed for the unbound enzyme (94% after 28 days). Similar trends were observed for encapsulated ENPs and unbound enzyme. Nevertheless, storage at −26°C resulted in an almost complete loss of enzymatic activity for all samples.

Complete wastewater discoloration by a novel peroxidase source with promising bioxidative properties

BACKGROUND Our study aimed to characterize and prospect immobilization strategies for a novel fungal peroxidase - POD (EC 1.11.1.7) and to insert it in the context of pollutant remediation, since these compounds pose risks to human and environmental health. The enzymatic extract was obtained by submerged fermentation of the fungus Trichoderma koningiopsis in an alternative substrate, consisting of fresh microalgal biomass. The immobilization efficiency was evaluated by monitoring the residual activity (RA) and the discoloration potential (DP) of a synthetic dye solution. Concomitantly, the catalytic properties of free POD were explored, and the most promising storage strategy to maintain the enzymatic activity was studied. RESULTS The novel guaiacol peroxidase expressed specific activity of up to 7801 U mg−1 in the free form, showing stability when subjected to up to 80°C in a pH range between 4.0-8.0. Furthermore, the bioproduct immobilized on magnetic nanoparticles expressed up to 689% RA and 100% DP. An increase in the RA of the enzyme, both in free and immobilized form, was also observed after storage for up to 8 months. The synthesized magnetic nanozymes showed good reusability, maintaining 13546 U mg−1 after ten cycles and removing 94% of color in a second batch. Toxicological evaluation with Allium cepa indicated that the enzymatic process of color removal with immobilized POD was essential to eliminate genotoxic effects. CONCLUSION T. koningiopsis peroxidase production and immobilization presented in our work are promising for the enzyme market and for the wastewater treatment technologies, considering its high bioxidative potential.

Exon-skipping antisense oligonucleotides for cystic fibrosis therapy

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site–targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon–containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

The Stability Improvement of α-Amylase Enzyme from Aspergillus fumigatus by Immobilization on a Bentonite Matrix

The stability of the α-amylase enzyme has been improved from Aspergillus fumigatus using the immobilization method on a bentonite matrix. Therefore, this study aims to obtain the higher stability of α-amylase enzyme from A. fumigatus; hence, it is used repeatedly to reduce industrial costs. The procedures involved enzyme production, isolation, partial purification, immobilization, and characterization. Furthermore, the soluble enzyme was immobilized using 0.1 M phosphate buffer of pH 7.5 on a bentonite matrix, after which it was characterized with the following parameters such as optimum temperature, Michaelis constant (KM), maximum velocity V max , thermal inactivation rate constant (ki), half-life (t1/2), and the change of energy due to denaturation (ΔGi). The results showed that the soluble enzyme has an optimum temperature of 55°C, KM of 3.04 mg mL−1 substrate, V max of 10.90 μmole mL−1 min−1, ki of 0.0171 min−1, t1/2 of 40.53 min, and ΔGi of 104.47 kJ mole−1, while the immobilized enzyme has an optimum temperature of 70°C, KM of 8.31 mg mL−1 substrate, V max of 1.44 μmole mL−1 min−1, ki of 0.0060 min−1, t1/2 of 115.50 min, and ΔGi of 107.37 kJ mole−1. Considering the results, the immobilized enzyme retained 42% of its residual activity after six reuse cycles. Additionally, the stability improvement of the α-amylase enzyme by immobilization on a bentonite matrix, based on the increase in half-life, was three times greater than the soluble enzyme.

Heterologous Expression and Rational Design of l-asparaginase from Rhizomucor miehei to Improve Thermostability

l-asparaginase (EC 3.5.1.1) hydrolyzes l-asparagine to produce l-aspartate and ammonia and is widely found in microorganisms, plants, and some rodent sera. l-asparaginase used for industrial production should have good thermostability. We heterologously expressed l-asparaginase from Rhizomucor miehei, selected nine loci for site-directed mutagenesis by rational design, and obtained two mutants with significantly improved thermostability. The optimal temperature of mutants S302I and S302M was 50 °C. After incubating the mutant and wild-type enzymes at 45 °C for 35 h, the residual activity of the wild-type enzyme (WT) was only about 10%. In contrast, the residual activity of S302I and S302M was more than 50%. After combination mutagenesis, Bacillus subtilis 168-pMA5-A344E/S302I was constructed using the food-safe host strain B. subtilis 168. Additionally, a 5′ untranslated region (UTR) modification strategy was adopted to enhance the expression level of R. miehei-derived l-asparaginase in B. subtilis. In a 5-L fermenter scale-up experiment, the enzyme activity of recombinant B. subtilis 168-pMA5-UTR-A344E/S302I reached 521.9 U·mL−1 by fed-batch fermentation.

Enzymatic Disruption of Biofilms During Cheese Manufacturing: A Mini Review

Bacteria are capable of colonizing industrial processing surfaces creating biofilms on them which may adversely affect the quality and safety of products. Traditional cleaning-in-place (CIP) treatments using caustic and nitric acid solutions have been known to exhibit variable efficiency in eliminating biofilm bacteria. Here, we introduce enzymes as an alternative to traditional CIP treatments and discuss their mechanism of action against bacterial biofilms in cheese manufacturing. In addition, we discuss research gaps namely thermal stability, substrate specificity and residual activity of enzymes that may play a vital role in the selection of enzymes with optimal effectiveness against multi species biofilms. The outcome of this mini review will aid in the development of a novel and sustainable enzyme-based CIP treatment during cheese manufacturing in the future.