Improvement of combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase by functionalization of cross-linker for maltooligosaccharides synthesis.

Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase (CGTase) and maltogenic amylase (Mag1) from Bacillus lehensis G1 (Combi-CLEAs-CM) were successfully developed to synthesis maltooligosaccharides (MOS). Yet, the poor cross-linking performance between chitosan (cross-linker) and enzymes resulting low activity recovery and catalytic efficiency. In this study, we proposed the functionalization of cross-linkers with the integration of computational analysis to study the influences of different functional group on cross-linkers in combi-CLEAs development. From in-silico analysis, O-carboxymethyl chitosan (OCMCS) with the highest binding affinity toward both enzymes was chosen and showed alignment with the experimental result, in which OCMCS was synthesized as cross-linker to develop improved activity recovery of Combi-CLEAs-CM-ocmcs (74 %). The thermal stability and deactivation energy (205.86 kJ/mol) of Combi-CLEAs-CM-ocmcs were found to be higher than Combi-CLEAs-CM (192.59 kJ/mol). The introduction of longer side chain of carboxymethyl group led to a more flexible structure of Combi-CLEAs-CM-ocmcs. This alteration significantly reduced the Km value of Combi-CLEAs-CM-ocmcs by about 3.64-fold and resulted in a greater Kcat/Km (3.63-fold higher) as compared to Combi-CLEAs-CM. Moreover, Combi-CLEAs-CM-ocmcs improved the reusability with retained >50 % of activity while Combi-CLEAs-CM only 36.18 % after five cycles. Finally, maximum MOS production (777.46 mg/g) was obtained by Combi-CLEAs-CM-ocmcs after optimization using response surface methodology.

Theoretical insight of reactive oxygen species scavenging mechanism in lignin waste depolymerization products.

Apart from natural products and synthesis, phenolic compounds can be produced from the depolymerization of lignin, a major waste in biofuel and paper production. This process yields a plethora of aryl propanoid phenolic derivatives with broad biological activities, especially antioxidant properties. Due to its versatility, our study focuses on investigating the antioxidant mechanisms of several phenolic compounds obtained from renewable and abundant resources, namely, syringol (Hs), 4-allylsyringol (HAs), 4-propenylsyringol (HPns), and 4-propylsyringol (HPs). Employing the density functional theory (DFT) approach in conjunction with the QM-ORSA protocol, we aim to explore the reactivity of these compounds in neutralizing hydroperoxyl radicals in physiological and non-polar media. Kinetic and thermodynamic parameter calculations on the antioxidant activity of these compounds were also included in this study. Additionally, our research utilizes the activation strain model (ASM) for the first time to explain the reactivity of the HT and RAF mechanisms in the peroxyl radical scavenging process. It is predicted that HPs has the best rate constant in both media (1.13 × 108 M-1 s-1 and 1.75 × 108 M-1 s-1, respectively). Through ASM analysis, it is observed that the increase in the interaction energy due to the formation of intermolecular hydrogen bonds during the reaction is an important feature for accelerating the hydrogen transfer process. Furthermore, by examining the physicochemical and toxicity parameters, only Hs is not suitable for further investigation as a therapeutic agent because of potential toxicity and mutagenicity. However, overall, all compounds are considered potent HOO˙ scavengers in lipid-rich environments compared to previously studied antioxidants.

Innovative biocatalyst synthesis of pectinolytic enzymes by cross-linking strategy: Potentially immobilised pectinases for the production of pectic-oligosaccharides from pectin.

Pectinases are outstanding multienzymes, which have the potential to produce new emerging pectic-oligosaccharides (POS) via enzymatic hydrolysis of pectin. However, free pectinase is unable to undergo repeated reaction for the production of POS. This study proposed a sustainable biocatalyst of pectinases known as cross-linked pectinase aggregates (CLPA). Pectinase from Aspergillus aculeatus was successfully precipitated using 2 mg/mL pectinase and 60 % acetone for 20 min at 20 °C, which remained 36.3 % of its initial activity. The prepared CLPA showed the highest activity recovery (85.0 %), under the optimised conditions (0.3 % (v/v) starch and glutaraldehyde mixture (St/Ga), 1.5: 1 of St/Ga, 25 °C, 1.5 h). Furthermore, pectin-degrading enzymes from various sources were used to produce different CLPA. The alteration of pectinase secondary structure gave high stability in acidic condition (pH 4), thermostability, deactivation energy and half-life, and improved storage stability at 4 °C for 30 days. Similarly to their free counterpart, the CLPA exhibited comparable enzymatic reaction kinetics and could be reused eight times with approximately 20 % of its initial activity. The developed CLPA does not only efficaciously produced POS from pectin as their free form, but also exhibited better operational stability and reusability, making it more suitable for POS production.

Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase from Bacillus lehensis G1 for maltooligosaccharides synthesis.

Maltooligosaccharides (MOS) are functional oligosaccharides that can be synthesized through enzymatic cascade reaction between cyclodextrin glucanotransferase (CGTase) and maltogenic amylase (Mag1) from Bacillus lehensis G1. To address the problems of low operational stability and non-reusability of free enzymes, both enzymes were co-immobilized as combined cross-linked enzyme aggregates (Combi-CLEAs-CM) with incorporation of bovine serum albumin (BSA) and Tween 80 (Combi-CLEAs-CM-add). Combi-CLEAs-CM and Combi-CLEAs-CM-add showed activity recoveries of 54.12 % and 69.44 %, respectively after optimization. Combi-CLEAs-CM-add showed higher thermal stability at higher temperatures (40 °C) with longer half-life (46.20 min) as compared to those of free enzymes (36.67 min) and Combi-CLEAs-CM (41.51 min). Both combi-CLEAs also exhibited higher pH stability over pH 5 to pH 9, and displayed excellent reusability with >50 % of initial activity retained after four cycles. The reduction in Km value of about 22.80 % and 1.76-fold increase in starch hydrolysis in comparison to Combi-CLEAs-CM attested the improvement of enzyme-substrate interaction by Tween 80 and pores formation by BSA in Combi-CLEAs-CM-add. The improved product specificity of Combi-CLEAs-CM-add also produced the highest yield of MOS (492 mg/g) after 3 h. Therefore, Combi-CLEAs-CM-add with ease of preparation, excellent reusability and high operational stability is believed to be highly efficacious biocatalyst for MOS production.

Antagonistic antimalarial properties of a methoxyamino chalcone derivative and 3-hydroxypyridinones in combination with dihydroartemisinin against Plasmodium falciparum.

Background: The spread of artemisinin (ART)-resistant Plasmodium falciparum threatens the control of malaria. Mutations in the propeller domains of P. falciparum Kelch13 (k13) are strongly associated with ART resistance. Ferredoxin (Fd), a component of the ferredoxin/NADP+ reductase (Fd/FNR) redox system, is essential for isoprenoid precursor synthesis in the plasmodial apicoplast, which is important for K13-dependent hemoglobin trafficking and ART activation. Therefore, Fd is an antimalarial drug target and fd mutations may modulate ART sensitivity. We hypothesized that loss of Fd/FNR function enhances the effect of k13 mutation on ART resistance. Methods: In this study, methoxyamino chalcone (C3), an antimalarial compound that has been reported to inhibit the interaction of recombinant Fd and FNR proteins, was used as a chemical inhibitor of the Fd/FNR redox system. We investigated the inhibitory effects of dihydroartemisinin (DHA), C3, and iron chelators including deferiprone (DFP), 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) and deferiprone-resveratrol hybrid (DFP-RVT) against wild-type (WT), k13 mutant, fd mutant, and k13 fd double mutant P. falciparum parasites. Furthermore, we investigated the pharmacological interaction of C3 with DHA, in which the iron chelators were used as reference ART antagonists. Results: C3 showed antimalarial potency similar to that of the iron chelators. As expected, combining DHA with C3 or iron chelators exhibited a moderately antagonistic effect. No differences were observed among the mutant parasites with respect to their sensitivity to C3, iron chelators, or the interactions of these compounds with DHA. Discussion: The data suggest that inhibitors of the Fd/FNR redox system should be avoided as ART partner drugs in ART combination therapy for treating malaria.

Update on the Development of Toehold Switch-Based Approach for Molecular Diagnostic Tests of COVID-19.

A high volume of diagnostic tests is needed during the coronavirus disease 2019 (COVID-19) pandemic to obtain representative results. These results can help to design and implement effective policies to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis using current gold standard methods, i.e., real-time quantitative PCR (RT-qPCR), is challenging, especially in areas with limited trained personnel and health-related infrastructure. The toehold switch-based diagnostic system is a promising alternative method for detecting SARS-CoV-2 that has advantages such as inexpensive cost per testing, rapid, and highly sensitive and specific analysis. Moreover, the system can be applied to paper-based platforms, simplifying the distribution and utilization in low-resource settings. This review provides insight into the development of toehold switch-based diagnostic devices as the most recent methods for detecting SARS-CoV-2.

Characterization of SARS-CoV-2 East Java isolate, Indonesia.

Background: Incidents of SARS-CoV-2 in East Java increased steadily, and it became the second epicenter in Indonesia. The COVID-19 pandemic caused a dire multisectoral crisis all around the world. This study investigates and characterizes local isolates from East Java, Indonesia.   Methods: There were 54 patients suspected with SARS-COV-2 infection and 27 patients were COVID-19 positive. Virus isolates were obtained from COVID-19 inpatients' nasopharyngeal swabs at the Dr Soetomo Teaching Hospital, Surabaya. There were only three isolates (#6, #11, #35) with good growth characteristics. Serial blind passage and cytopathic effect observation in the Vero E6 cell line were performed for virus isolation. Confirmation of the SARS-CoV-2 infection was proven by means of reverse transcriptase-polymerase chain reactions using SARS-CoV-2 specific primers, scanning electron microscopy, and scanning transmission electron microscopy examination. Whole genome sequencing was performed using ARTIC protocol. Furthermore, SARS-CoV-2 characterization was identified through a western blot using rabbit serum immunized with inactive SARS-CoV-2 vaccine and human natural COVID-19 infection serum.   Results: Spike gene analysis of three samples (#6, #11, #35) found that the D614G mutation was detected in all isolates, although one isolate exhibited the D215Y and E484D mutation. Based on whole genome analysis, those three isolates were included in clade 20A, and two isolates were included in lineage B.1.6 with one isolate belongs to lineage B.1.4.7.   Conclusion: Based on molecular characterization and immunogenicity of SARS-CoV-2 East Java, Indonesia showed high titer and it has mutation in some regions.

In silico characterization of the GH5-cellulase family from uncultured microorganisms: physicochemical and structural studies.

BACKGROUND: Hydrolysis of cellulose-based biomass by cellulases produce fermented sugar for making biofuels, such as bioethanol. Cellulases hydrolyze the ß-1,4-glycosidic linkage of cellulose and can be obtained from cultured and uncultured microorganisms. Uncultured microorganisms are a source for exploring novel cellulase genes through the metagenomic approach. Metagenomics concerns the extraction, cloning, and analysis of the entire genetic complement of a habitat without cultivating microbes. The glycoside hydrolase 5 family (GH5) is a cellulase family, as the largest group of glycoside hydrolases. Numerous variants of GH5-cellulase family have been identified through the metagenomic approach, including CelGH5 in this study. University-CoE-Research Center for Biomolecule Engineering, Universitas Airlangga successfully isolated CelGH5 from waste decomposition of oil palm empty fruit bunches (OPEFB) soil by metagenomics approach. The properties and structural characteristics of GH5-cellulases from uncultured microorganisms can be studied using computational tools and software. RESULTS: The GH5-cellulase family from uncultured microorganisms was characterized using standard computational-based tools. The amino acid sequences and 3D-protein structures were retrieved from the GenBank Database and Protein Data Bank. The physicochemical analysis revealed the sequence length was roughly 332-751 amino acids, with the molecular weight range around 37-83 kDa, dominantly negative charges with pI values below 7. Alanine was the most abundant amino acid making up the GH5-cellulase family and the percentage of hydrophobic amino acids was more than hydrophilic. Interestingly, ten endopeptidases with the highest average number of cleavage sites were found. Another uniqueness demonstrated that there was also a difference in stability between in silico and wet lab. The II values indicated CelGH5 and ACA61162.1 as unstable enzymes, while the wet lab showed they were stable at broad pH range. The program of SOPMA, PDBsum, ProSA, and SAVES provided the secondary and tertiary structure analysis. The predominant secondary structure was the random coil, and tertiary structure has fulfilled the structure quality of QMEAN4, ERRAT, Ramachandran plot, and Z score. CONCLUSION: This study can afford the new insights about the physicochemical and structural properties of the GH5-cellulase family from uncultured microorganisms. Furthermore, in silico analysis could be valuable in selecting a highly efficient cellulases for enhanced enzyme production.

Initial study on TMPRSS2 p.Val160Met genetic variant in COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a global health problem that causes millions of deaths worldwide. The clinical manifestation of COVID-19 widely varies from asymptomatic infection to severe pneumonia and systemic inflammatory disease. It is thought that host genetic variability may affect the host's response to the virus infection and thus cause severity of the disease. The SARS-CoV-2 virus requires interaction with its receptor complex in the host cells before infection. The transmembrane protease serine 2 (TMPRSS2) has been identified as one of the key molecules involved in SARS-CoV-2 virus receptor binding and cell invasion. Therefore, in this study, we investigated the correlation between a genetic variant within the human TMPRSS2 gene and COVID-19 severity and viral load. RESULTS: We genotyped 95 patients with COVID-19 hospitalised in Dr Soetomo General Hospital and Indrapura Field Hospital (Surabaya, Indonesia) for the TMPRSS2 p.Val160Met polymorphism. Polymorphism was detected using a TaqMan assay. We then analysed the association between the presence of the genetic variant and disease severity and viral load. We did not observe any correlation between the presence of TMPRSS2 genetic variant and the severity of the disease. However, we identified a significant association between the p.Val160Met polymorphism and the SARS-CoV-2 viral load, as estimated by the Ct value of the diagnostic nucleic acid amplification test. Furthermore, we observed a trend of association between the presence of the C allele and the mortality rate in patients with severe COVID-19. CONCLUSION: Our data indicate a possible association between TMPRSS2 p.Val160Met polymorphism and SARS-CoV-2 infectivity and the outcome of COVID-19.

Effect of Poly-Histidine Tag Position toward Inhibition Activity of Secretory Leukocyte Protease Inhibitor as Candidate for Material Wound Healing.

BACKGROUND: The Secretory Leukocyte Protease Inhibitors (SLPI) has many biological functions including anti-bacterial, anti-fungal, anti-viral, anti-inflammatory, and immuno-modulatory. Previous studies have shown that gene-encoding human SLPI have successfully been expressed in Escherichia coli (E. coli) with a C-terminal poly-histidine tag (His-tag). The aim of this research was to investigate the inhibition activity of N-terminal His-tag position (NSLPI) and C-terminal His-tag position (CSLPI). We hypothesized that a His-tag close to an active site SLPI domain may interfere with the inhibition activity of SLPIs. METHODS: A NSLPI and CSLPI were constructed with polymerase chain reaction (PCR) amplification. The PCR products were then ligated into pET-30a plasmid and transformed into E. coli TOP10. Recombinant plasmids were verified by using restriction analysis and nucleotide sequence analysis. pET-NSLPI and pET-CSLPI were then subcloned in E. coli BL21(DE3) for its expression. The SLPI protein was expressed using Isopropyl ß-D-1-thiogalactopyranoside induction (IPTG). The inhibition effect of both SLPI against Porcine Pancreatic Elastase (PPE) enzyme was tested using the N-succinyil-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-4-nitroanalide (NPN) substrate. RESULTS: The SLPI gene was successfully cloned and expressed in E. coli BL21. Fusion proteins of NSLPI and CSLPI were generated with His-tag in the N-terminal and C-terminal position, respectively. The inhibition effect of NSLPI and CSLPI on PPE indicated that both SLPI were active. The inhibition activity of NSLPI was 66.7%, higher than CSLPI by 44.4%. CONCLUSION: The His-tag position on the C-terminal of SLPI reduced the inhibition activity of SLPI.

ß-Xylosidases: Structural Diversity, Catalytic Mechanism, and Inhibition by Monosaccharides.

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, ß-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the ß-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, ß-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of ß-xylosidases, and discuss their inhibition by monosaccharides.

Structural basis of product inhibition by arabinose and xylose of the thermostable GH43 ß-1,4-xylosidase from Geobacillus thermoleovorans IT-08.

Complete degradation of the xylan backbone of hemicellulosic plant cell walls requires the synergistic action of endo-xylanases and ß-1,4-xylosidases. While endo-xylanases produce xylooligosaccharides from xylan, ß-1,4-xylosidases degrade the xylooligosaccharides into xylose monomers. The glycoside hydrolase family 43 ß-1,4-xylosidase from Geobacillus thermoleovorans IT-08 is a promising, heat stable catalyst for the saccharification of hemicellulosic material into simple fermentable sugars, but it is competitively inhibited by its products arabinose and xylose. As a first step to help overcome this problem, we elucidated crystal structures of the enzyme in the unliganded form and with bound products, at 1.7-2.0 Å resolution. The structures are very similar to those of other enzymes belonging to glycoside hydrolase family 43. Unexpectedly, the monosaccharides are bound in very different ways. Arabinose preferentially binds in subsite -1, while xylose exclusively interacts with subsite +1. These structures and sugar binding preferences suggest ways for improving the catalytic performance of the enzyme by rational mutational design.

Design and synthesis of chalcone derivatives as inhibitors of the ferredoxin - ferredoxin-NADP+ reductase interaction of Plasmodium falciparum: pursuing new antimalarial agents.

Some chalcones have been designed and synthesized using Claisen-Schmidt reactions as inhibitors of the ferredoxin and ferredoxin-NADP+ reductase interaction to pursue a new selective antimalaria agent. The synthesized compounds exhibited inhibition interactions between PfFd-PfFNR in the range of 10.94%-50%. The three strongest inhibition activities were shown by (E)-1-(4-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (50%), (E)-1-(4-aminophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (38.16%), and (E)-1-(4-aminophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-1-one (31.58%). From the docking experiments we established that the amino group of the methoxyamino chlacone derivatives plays an important role in the inhibition activity by electrostatic interaction through salt bridges and that it forms more stable and better affinity complexes with FNR than with Fd.

ß-D-xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme.

The gene encoding a thermostable ß-D-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6-8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s(-1) mM(-1) on p-nitrophenyl-ß-D-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel ß-strands) and catalytic module (residues 157 to 604 forming five-bladed ß-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08.

The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-beta-xylanase and beta-xylosidase. beta-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-beta-xylanase into xylose monomers. The beta-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4(3)2(1)2, with unit-cell parameters a = b = 62.53, c = 277.4 A diffracted to 1.55 A resolution. The rectangular crystals belonged to space group P2(1), with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 A, beta = 90.5 degrees , and diffracted to 1.80 A resolution.