Data on gene cloning, expression, purification, and characterization of the glycoside hydrolase family 11 from Bacillus velezensis.

Bacillus velezensis RB.IBE29 is a chitinolytic bacterium originally isolated from the rhizospheric soil of black pepper grown in Vietnam. This bacterium is a strong biocontrol agent against plant pathogens and possesses a novel chitinase system. Genome sequences available in CAZy database revealed B. velezensis possesses one gene encoding xylanase belonging to glycoside hydrolase family 11; however, this enzyme has yet to be un-experimentally characterized. In this work, xyA gene was isolated from the genomic DNA of strain RB.IBE29 and cloned in Escherichia coli DH5α cells using the pUC19 vector. Sequencing analysis showed that the ORF of xyA contains 642 bp and encodes the deduced xylanase with 213 aa and 23.27 kDa. The domain structure of the enzyme has a signal peptide and a family 11 catalytic domain. xyA (without peptide sequence) was successfully expressed in E. coli BL21-CodonPlus (DE3)-RIPL cells using the pColdII vector and purified using the HisTrap FF column. Purified recombinant xylanase degraded xylan substrates, had the highest hydrolytic activity at 55°C in 20 mM sodium phosphate buffer (pH 6.0), and MgCl2, CoCl2, and MnCl2 enhanced the enzymatic activity. Nucleotide sequence of xyA was submitted to the DDBJ/GenBank/EMBL under accession number LC779040. This is the first data on the gene cloning, expression, purification, and characterization of the glycoside hydrolase family 11 from B. velezensis.

Complete genome sequence data of chitin-degrading Bacillus velezensis RB.IBE29.

This work reports the complete genome sequence of chitinolytic Bacillus velezensis RB.IBE29 recently isolated from the rhizosphere of black pepper cultivated in the Central Highlands region of Vietnam. This bacterium had strong antagonistic activity against phytopathogens and possessed a novel chitinase system. The complete genome of strain RB.IBE29 was sequenced using the platforms of Illumina (2×150 PE) and Oxford Nanopore technologies. Assembly showed that strain RB.IBE29 has one 3,957,092-bp circular chromosome with 46.5 % G+C content. DFAST analysis revealed the genome contains 3819 protein-coding genes, 27 rRNAs, 86 tRNAs, 1 tmRNA, 144 pseudogenes, and shares an ANI value of 97.51 % with that of reported B. velezensis NRRL B-41580. The B. velezensis RB.IBE29 genome possesses at least 42 genes concerning heavy metal resistance and plant-growth promotion. CAZymes analysis showed that 103 genes coding for carbohydrate-active enzymes were predicted in the genome, including 41 genes for glycoside hydrolases, 34 genes for glycosyl transferases, 3 genes for polysaccharide lyases, 17 genes for carbohydrate esterases, 6 genes for auxiliary activities, and 2 genes for carbohydrate-binding modules. Of these deduced enzymes, at least 8 probably possess activities against phytopathogens, such as family 18 chitinases, family 16 glucanase, and family 46 chitosanase. AntiSMASH analysis exhibited that 15 biosynthetic gene clusters were found in the genome; among them, 5 show no sequence similarity to known bacterial clusters. The raw sequences in this work were deposited in Mendeley Data. The complete genome sequence of strain RB.IBE29 was submitted to the DDBJ/GenBank/EMBL under accession number AP028932. The obtained data provide insight into the biocontrol ability and plant-growth promotion of B. velezensis RB.IBE29. The data are valuable for further explorations concerning crop production and other fields using gene expression approaches.

Assessment of the Chemical Profile and Potential Medical Effects of a Flavonoid-Rich Extract of Eclipta prostrata L. Collected in the Central Highlands of Vietnam.

Eclipta prostrata L. (EPL), a medicinal plant, is widely utilized in the central highlands of Vietnam. This study aims to assess the chemical profile and potential medical effects of an EPL extract rich in flavonoids. A total of 36 secondary metabolites were identified from the EPL extract through GC-MS and UHPLC-UV analysis. Among them, 15 volatile compounds and several phenolic and flavonoid chemicals, including salicylic acid, epicatechin gallate, isovitexin, and apigetrin, were reported in EPL extract for the first time. This herbal extract demonstrated moderate inhibition against α-amylase and α-glucosidase, and high anti-oxidant and anti-acetylcholinesterase activities (IC50 = 76.8 ± 0.8 µg/mL). These promising attributes can be likely attributed to the high levels of major compounds, including wedelolactone (1), chlorogenic acid (3), epicatechin gallate (6), salicylic acid (8), isovitexin (9), apigetrin (11), and myricetin (12). These findings align with the traditional use of EPL for enhancing memory and cognitive function, as well as its potential benefits in diabetes management. The results of the molecular docking study reveal that the major identified compounds (1, 6, 9, and 11) showed a more effective acetylcholinesterase inhibitory effect than berberine chloride, with good binding energy (DS values, -12.3 to -14.3 kcal/mol) and acceptable values of RMSD (1.02-1.67 Å). Additionally, almost all the identified major compounds exhibited good ADMET properties within the required limits.

Chitosanase Production from the Liquid Fermentation of Squid Pens Waste by Paenibacillus elgii.

Chitosanases play a significant part in the hydrolysis of chitosan to form chitooligosaccharides (COS) that possess diverse biological activities. This study aimed to enhance the productivity of Paenibacillus elgii TKU051 chitosanase by fermentation from chitinous fishery wastes. The ideal parameters for achieving maximum chitosanase activity were determined: a squid pens powder amount of 5.278% (w/v), an initial pH value of 8.93, an incubation temperature of 38 °C, and an incubation duration of 5.73 days. The resulting chitosanase activity of the culture medium was 2.023 U/mL. A chitosanase with a molecular weight of 25 kDa was isolated from the culture medium of P. elgii TKU051 and was biochemically characterized. Liquid chromatography with tandem mass spectrometry analysis revealed that P. elgii TKU051 chitosanase exhibited a maximum amino acid identity of 43% with a chitosanase of Bacillus circulans belonging to the glycoside hydrolase (GH) family 46. P. elgii TKU051 chitosanase demonstrated optimal activity at pH 5.5 while displaying remarkable stability within the pH range of 5.0 to 9.0. The enzyme displayed maximum efficiency at 60 °C and demonstrated considerable stability at temperatures ≤40 °C. The presence of Mn2+ positively affected the activity of the enzyme, while the presence of Cu2+ had a negative effect. Thin-layer chromatography analysis demonstrated that P. elgii TKU051 chitosanase exhibited an endo-type cleavage pattern and hydrolyzed chitosan with 98% degree of deacetylation to yield (GlcN)2 and (GlcN)3. The enzymatic properties of P. elgii TKU051 chitosanase render it a promising candidate for application in the production of COS.

Novel resources of chitinolytic bacteria isolated from Yok Don National Park, Vietnam.

AIMS: This study focused on the isolation and characterization of chitinolytic bacteria from Yok Don National Park, Vietnam for future studies regarding biofertilizers and biocontrol agents. METHODS AND RESULTS: Chitinolytic bacteria were isolated from soils and chitin flakes soaked in river water at the National Park. On the basis of the halo zones caused by colloidal chitin degradation and colony morphologies, 12 chitinolytic strains were chosen from 15 700 isolates for various examinations. Findings from 16S rDNA analysis indicated that among these strains, 10 could be identified as different species, and the remaining 2 showed less identity to known species and genera. The 12 bacteria possess numerous properties concerning plant growth promotion and/or phytopathogenic biocontrol. Paenibacillus chitinolyticus YSY-3.1, which exhibited the highest chitinase activity and remarkable properties for plant growth, was chosen for sequencing and draft genome analysis. The results showed that the genome is 6571 781 bp in length with 6194 coding sequences, 52.2% G + C, and 96.53% ANI value. It harbors the chitinolytic system comprising 22 enzymes. Among these enzymes, PcChiQ has a loop structure different from that of known family 19 chitinases, PcChiA contains two GH18 catalytic domains rarely found in microorganisms, and PcChiF contains three GH18 catalytic domains that have never been reported. CONCLUSIONS: The 12 identified chitinolytic bacteria exhibit great potential for further studies on plant growth-promoting and/or biocontrol properties. Among these bacteria, two strains might be good candidates for next examinations concerning novel species and/or genera, and strain YSY-3.1 could possess a novel chitinolytic system.

Novel Anti-Acetylcholinesterase Effect of Euonymus laxiflorus Champ. Extracts via Experimental and In Silico Studies.

Alzheimer's disease (AD) is the most common form of dementia, which is recorded as a global health issue. Natural acetylcholinesterase inhibitors (AChEIs) are considered a helpful therapy for the management of symptoms of patients with mild-to-moderate AD. This work aimed to investigate and characterize Euonymus laxiflorus Champ. (ELC) as a natural source of AChEIs compounds via in vitro and virtual studies. The screening parts used, including the leaves, heartwood, and trunk bark of ELC, revealed that the trunk bark extract possessed the highest activity, phenolics and flavonoid content. The in vitro anti-Alzheimer activity of ELC trunk bark was notably reclaimed for the first time with comparable effect (IC50 = 0.332 mg/mL) as that of a commercial AChEI, berberine chloride (IC50 = 0.314 mg/mL). Among various solvents, methanol was the most suitable to extract ELC trunk bark with the highest activity. Twenty-one secondary metabolites (1-21) were identified from ELC trunk bark extract, based on GCMS and UHPLC analyses. Of these, 10 volatile compounds were identified from this herbal extract for the first time. One phenolic (11) and seven flavonoid compounds (15-21) were also newly found in this herbal extract. Of the identified compounds, chlorogenic acid (11), epigallocatechin gallate (12), epicatechin (13), apigetrin (18), and quercetin (20) were major compounds with a significant content of 395.8-2481.5 µg/g of dried extract. According to docking-based simulation, compounds (11-19, and 21) demonstrated more effective inhibitory activity than berberine chloride, with good binding energy (DS values: -12.3 to -14.4 kcal/mol) and acceptable RMSD values (0.77-1.75 Å). In general, these identified compounds processed drug properties and were non-toxic for human use, based on Lipinski's rule of five and ADMET analyses.

Screening and Elucidation of Chemical Structures of Novel Mammalian α-Glucosidase Inhibitors Targeting Anti-Diabetes Drug from Herbals Used by E De Ethnic Tribe in Vietnam.

Among ten extracts of indigenous medicinal plants, the MeOH extract of Terminalia triptera Stapf. (TTS) showed the most efficient mammalian α-glucosidase inhibition for the first time. The data of screening bioactive parts used indicated that the TTS trunk bark and leaves extracts demonstrated comparable and higher effects compared to acarbose, a commercial anti-diabetic drug, with half-maximal inhibitory concentration (IC50) values of 181, 331, and 309 µg/mL, respectively. Further bioassay-guided purification led to the isolation of three active compounds from the TTS trunk bark extract and identified as (-)-epicatechin (1), eschweilenol C (2), and gallic acid (3). Of these, compounds 1 and 2 were determined as novel and potent mammalian α-glucosidase inhibitors. The virtual study indicated that these compounds bind to α-glucosidase (Q6P7A9) with acceptable RMSD values (1.16-1.56 Å) and good binding energy (DS values in the range of -11.4 to -12.8 kcal/mol) by interacting with various prominent amino acids to generate five and six linkages, respectively. The data of Lipinski's rule of five and absorption, distribution, metabolism, excretion and toxicity (ADMET)-based pharmacokinetics and pharmacology revealed that these purified compounds possess anti-diabetic drug properties, and the compounds are almost not toxic for human use. Thus, the findings of this work suggested that (-)-epicatechin and eschweilenol C are novel potential mammalian α-glucosidase inhibitor candidates for type 2 diabetes treatment.

Metagenomic next-generation sequencing of the microbiome dataset from the surface water sample collected from Serepok River in Yok Don National Park, Vietnam.

The Central Highlands region is considered as the center with the highest biodiversity in Vietnam because it has the majority of national parks such as Yok Don, Chu Yang Sin, Bidoup-Nui Ba, Ta Dung, Chu Mon Ray, and Kon Ka Kinh and nature reserves such as Ngoc Linh, Kon Chu Rang, Ea So, Nam Ka, and Nam Nung with different ecosystems [1]. Of the national parks and nature reserves, Yok Don has the most different ecosystem. Yok Don is the second biggest national park, and it is the only national park that conserves dry deciduous dipterocarp forests in Vietnam [2]. Presently, the decrease in forest area and global warming have led to the continuous reduction in microbial resources in this region. Thus, a dataset of the soil microbiome in this region has been established to explore microbial resources for conservation and further application in sustainable agricultural production in this region [3]; however, to the best of our knowledge, a dataset of water microbiome remains unknown. This work presented a microbiome dataset from surface water samples collected from Serepok River in Yok Don National Park, Vietnam. Metagenomic next-generation sequencing was used to characterize microbial communities in the sample. The raw sequence in this work was uploaded in Fastq format on NCBI, which can be accessed at https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA853090. This metagenome dataset can provide valuable information on surface water microbial communities and their functionality. It can also be used for further studies on the conservation and application of indigenous microbial resources for sustainable crop production in this region.

Potential of the Liquid Fermentation of Fishery Waste by Paenibacillus elgii for Metalloprotease Production.

This study attempted to use fishery processing wastes to produce protease by Paenibacillus elgii TKU051. Of the tested wastes, tuna head powder (THP) was found to be the most effective carbon and nitrogen (C/N) source, and the optimal conditions were as follows: 0.811% THP, 0.052% K2HPO4, 0.073% MgSO4, initial pH of 8.96, incubation temperature of 31.4 °C, and incubation time of 3.092 days to achieve the maximum protease activity of 2.635 ± 0.124 U/mL. A protease with a molecular weight of 29 kDa was purified and biochemically characterized. Liquid chromatography with tandem mass spectrometry analysis revealed an amino acid sequence of STVHYSTR of P. elgii TKU051 protease, suggesting that the enzyme may belong to the M4 family of metalloproteases. The optimal activity of the enzyme was achieved at 60 °C and pH 8. P. elgii TKU051 protease was strongly inhibited by ethylenediaminetetraacetic acid and 1,10-phenanthroline, indicating its precise metalloprotease property. P. elgii TKU051 protease displayed the activity toward casein and raw fishery wastes such as tuna heads, tuna viscera, shrimp heads, and squid pens. Finally, the purified P. elgii TKU051 protease could improve the free-radical scavenging activity of fishery wastes. In short, P. elgii TKU051 has potential application in eco-friendly approaches to efficiently convert fishery wastes to metalloprotease.

Novel α-Amylase Inhibitor Hemi-Pyocyanin Produced by Microbial Conversion of Chitinous Discards.

α-Amylase inhibitors (aAIs) have been applied for the efficient management of type 2 diabetes. The aim of this study was to search for potential aAIs produced by microbial fermentation. Among various bacterial strains, Pseudomonas aeruginosa TUN03 was found to be a potential aAI-producing strain, and shrimp heads powder (SHP) was screened as the most suitable C/N source for fermentation. P. aeruginosa TUN03 exhibited the highest aAIs productivity (3100 U/mL) in the medium containing 1.5% SHP with an initial pH of 7-7.5, and fermentation was performed at 27.5 °C for two days. Further, aAI compounds were investigated for scaled-up production in a 14 L-bioreactor system. The results revealed a high yield (4200 U/mL) in a much shorter fermentation time (12 h) compared to fermentation in flasks. Bioactivity-guided purification resulted in the isolation of one major target compound, identified as hemi-pyocyanin (HPC) via gas chromatography-mass spectrometry and nuclear magnetic resonance. Its purity was analyzed by high-performance liquid chromatography. HPC demonstrated potent α-amylase inhibitory activity comparable to that of acarbose, a commercial antidiabetic drug. Notably, HPC was determined as a new aAI. The docking study indicated that HPC inhibits α-amylase by binding to amino acid Arg421 at the biding site on enzyme α-amylase with good binding energy (-9.3 kcal/mol) and creating two linkages of H-acceptors.

Analysis of endophytic microbiome dataset from roots of black pepper (Piper nigrum L.) cultivated in the Central Highlands region, Vietnam using 16S rRNA gene metagenomic next-generation sequencing.

Vietnam is the most prominent black pepper producer and exporter in the world. In 2020, the cultivated area of black pepper in Vietnam was 132.000 hectares and its production was 270.000 tons, in which the Central Highlands region took about 70% of both the cultivated area and production [1]. Hence, this region is thought to be the capital of black pepper cultivation and production in Vietnam. Numerous researches have investigated biodiversity and collected various beneficial endophytic bacteria from this plant in this region; however, traditional methods only were used to isolate such bacteria [2], [3], [4], [5]. Therefore, these studies have a limitation to providing insight into the profiles of the endophytic microorganism dataset in the black pepper plant. Most recently, our work based on the 16S rRNA gene amplicon sequencing revealed an insight into profiles of microbial diversity and its functionality from the sample collected from a forest in this region; however, that work was just focused on soil microbiome dataset from the dry deciduous dipterocarp forest in Yok Don national park [6]. To our knowledge, a dataset of endophytic microbiome of black pepper plant cultivated in the Central Highlands remains unclear. This report presents a dataset of the endophytic microbiome from a representative sample combined from five different root samples of black pepper (Vinh Linh local variety) cultivated in the Central Highlands of Vietnam using 16S rRNA gene metagenomic next-generation sequencing. The dataset in this work can provide information on the endophytic microbial diversity and its functionality. It can also be useful for developing cultivation techniques by applying endophytic microbial genetic resources for sustainable black pepper production in the Central Highlands, Vietnam, towards the nutrient need in different stages of development and growth.

Expression, purification, and basic properties of a novel domain structure possessing chitinase from Escherichia coli carrying the family 18 chitinase gene of Bacillus velezensis strain RB.IBE29.

BACKGROUND: Bacillus velezensis possesses numerous chitinolytic enzymes; however, not much is known about the role of its chitinase molecules. METHODS AND RESULTS: In this study, the chiA gene, which encodes a novel domain structure possessing family 18 chitinase from B. velezensis RB.IBE29, was expressed successfully in Escherichia coli BL21-CodonPlus (DE3)-RIPL using the pColdII expression vector. The recombinant protein, rBvChiA, was purified using the HisTrap FF column. Purified rBvChiA showed hydrolytic activity against insoluble chitin and bound to chitinous substrates. In addition, the purified recombinant enzyme displayed remarkable inhibition effects on the spore germination of Fusarium falciforme and the egg hatch of root-knot nematodes (Meloidogyne spp.), which are the main causes of black pepper diseases in the Central Highlands region, Vietnam. CONCLUSION: The current work results might enable further studies to develop novel chitinase A and strain RB.IBE29 as a natural fungicide and nematicide for sustainable black pepper production and other crops in the Central Highlands, Vietnam. This is the second report describing chitinase from B. velezensis based on the experimental data.

Soil microbiome dataset from Yok Don national park in the Central Highlands region of Vietnam.

The Central Highlands region contains most of the national parks in Vietnam with different ecosystems, including the national parks of Kon Ka Kinh, Chu Mon Ray, Chu Yang Sin, Yok Don, Bidoup-Nui Ba, and Ta Dung. Thus, this region is considered a center with the highest biodiversity in Vietnam [1]. Among the national parks, Yok Don is unique in its conservation of the dry deciduous dipterocarp forest. Furthermore, Yok Don is the second-largest park in Vietnam; it has the most different ecosystem compared with other national parks in this region [2]. Although some studies have investigated biodiversity preservation in the region, some other studies have only dealt with medicinal plants, lichens, and the rhizospheric bacteria of cultivated black pepper [1,[3], [4], [5]. To the best of our knowledge, no research on the microbial communities in Yok Don national park and in the Central Highlands has been reported. At present, global warming and a decrease in the forest area in the Central Highlands have led to the ongoing reduction in biodiversity and microbial resources. The current study reports the microbiome dataset from the soil sample collected in Yok Don national park. Metagenomic next-generation sequencing was used to characterize the microbial communities in the sample. The metagenome dataset generated provides information on microbial diversity and its functionality and can be useful for further studies on the conservation and use of microbial genetic resources in this region.

Molecular analysis of genes involved in chitin degradation from the chitinolytic bacterium Bacillus velezensis.

Bacillus velezensis RB.IBE29 is a potent biocontrol agent with high chitinase activity isolated from the rhizosphere of black pepper cultivated in the Central Highlands, Vietnam. Genome sequences revealed that this species possesses some GH18 chitinases and AA10 protein(s); however, these enzymes have not been experimentally characterized. In this work, three genes were identified from the genomic DNA of this bacterium and cloned in Escherichia coli. Sequence analysis exhibited that the ORF of chiA consists of 1,203 bp and encodes deduced 45.46 kDa-chitinase A of 400 aa. The domain structure of chitinase A is composed of a CBM 50 domain at the N-terminus and a catalytic domain at the C-terminus. The ORF of chiB includes 1,263 bp and encodes deduced 47.59 kDa-chitinase B of 420 aa. Chitinase B consists of two CBM50 domains at the N-terminus and a catalytic domain at the C-terminus. The ORF of lpmo10 is 621 bp and encodes a deduced 22.44 kDa-AA10 protein, BvLPMO10 of 206 aa. BvLPMO10 contains a signal peptide and an AA10 catalytic domain. Chitinases A and B were grouped into subfamily A of family 18 chitinases. Amino acid sequences in their catalytic domains lack aromatic residues (Trp, Phe, Tyr) probably involved in processivity and substrate binding compared with well-known bacterial GH18 chitinases. chiB was successfully expressed in E. coli. Purified rBvChiB degraded insoluble chitin and was responsible for inhibition of fungal spore-germination and egg hatching of plant-parasitic nematode. This is the first report describing the analysis of the chitinase system from B. velezensis.

Utilization of Cassava Wastewater for Low-Cost Production of Prodigiosin via Serratia marcescens TNU01 Fermentation and Its Novel Potent α-Glucosidase Inhibitory Effect.

The purpose of this study was to reuse cassava wastewater (CW) for scaled-up production, via the fermentation of prodigiosin (PG), and to conduct an evaluation of its bioactivities. PG was produced at the yield of high 6150 mg/L in a 14 L-bioreactor system, when the designed novel medium (7 L), containing CW and supplemented with 0.25% casein, 0.05% MgSO4, and 0.1% K2HPO4, was fermented with Serratia marcescens TNU01 at 28 °C in 8 h. The PG produced and purified in this study was assayed for some medical effects and showed moderate antioxidant, high anti-NO (anti-nitric oxide), and potential α-glucosidase inhibitory activities. Notably, PG was first reported as a novel effective α-glucosidase inhibitor with a low IC50 value of 0.0183 µg/mL. The commercial anti-diabetic drug acarbose was tested for comparison and had a lesser effect with a high IC50 value of 328.4 µg/mL, respectively. In a docking study, the cation form of PG (cation-PG) was found to bind to the enzyme α-glucosidase by interacting with two prominent amino acids, ASP568 and PHE601, at the binding site on the target enzyme, creating six linkages and showing a better binding energy score (-14.6 kcal/mol) than acarbose (-10.5 kcal/mol). The results of this work suggest that cassava wastewater can serve as a low-cost raw material for the effective production of PG, a potential antidiabetic drug candidate.

Proteases Production and Chitin Preparation from the Liquid Fermentation of Chitinous Fishery By-Products by Paenibacillus elgii.

Chitinous fishery by-products have great application in the production of various bioactive compounds. In this study, Paenibacillus elgii TKU051, a protease-producing bacterial strain, was isolated using a medium containing 1% squid pens powder (SPP) as the sole carbon/nitrogen (C/N) source. P. elgii TKU051 was found to produce at least four proteases with molecular weights of 100 kDa, 57 kDa, 43 kDa, and 34 kDa (determined by the gelatin zymography method). A P. elgii TkU051 crude enzyme cocktail was optimally active at pH 6-7 and 60 °C. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and α-glucosidase inhibitory activity of the hydrolysates obtained from the hydrolysis of shrimp shell powder, shrimp head powder, shrimp meat powder, fish head powder and soya bean powder catalyzed by the P. elgii TkU051 crude enzyme cocktail were also evaluated. P. elgii TKU051 exhibited a high deproteinization capacity (over 94%) on different kinds of shrimp waste (shrimp heads and shells; fresh and cooked shrimp waste; shrimp waste dried by oven and lyophilizer), and the Fourier-transform infrared spectroscopy profile of the chitin obtained from the deproteinization process displayed the characteristic of chitin. Finally, the obtained chitin exhibited an effect comparable to commercial chitin in terms of adsorption against Congo Red (90.48% and 90.91%, respectively). Thus, P. elgii TKU051 showed potential in the reclamation of chitinous fishery by-products for proteases production and chitin extraction.

Production of Sucrolytic Enzyme by Bacillus licheniformis by the Bioconversion of Pomelo Albedo as a Carbon Source.

Recently, there has been increasing use of agro-byproducts in microbial fermentation to produce a variety of value-added products. In this study, among various kinds of agro-byproducts, pomelo albedo powder (PAP) was found to be the most effective carbon source for the production of sucrose hydrolyzing enzyme by Bacillus licheniformis TKU004. The optimal medium for sucrolytic enzyme production contained 2% PAP, 0.75% NH4NO3, 0.05% MgSO4, and 0.05% NaH2PO4 and the optimal culture conditions were pH 6.7, 35 °C, 150 rpm, and 24 h. Accordingly, the highest sucrolytic activity was 1.87 U/mL, 4.79-fold higher than that from standard conditions using sucrose as the carbon source. The purified sucrolytic enzyme (sleTKU004) is a 53 kDa monomeric protein and belongs to the glycoside hydrolase family 68. The optimum temperature and pH of sleTKU004 were 50 °C, and pH = 6, respectively. SleTKU004 could hydrolyze sucrose, raffinose, and stachyose by attacking the glycoside linkage between glucose and fructose molecules of the sucrose unit. The Km and Vmax of sleTKU004 were 1.16 M and 5.99 µmol/min, respectively. Finally, sleTKU004 showed strong sucrose tolerance and presented the highest hydrolytic activity at the sucrose concentration of 1.2 M-1.5 M.

Conversion of Pectin-Containing By-Products to Pectinases by Bacillus amyloliquefaciens and Its Applications on Hydrolyzing Banana Peels for Prebiotics Production.

The utilization of pectin-containing by-products may be useful in a variety of fields. This study aims to establish the processing of pectin-containing by-products to produce pectinases using Bacillus amyloliquefaciens TKU050 strain. In this study, several kinds of agricultural pectin-containing by-products from banana (banana peel), rice (rice bran), orange (orange peel), coffee (spent coffee grounds), and wheat (wheat bran) were utilized to provide carbon sources for the production of a pectinase by B. amyloliquefaciens TKU050. B. amyloliquefaciens TKU050 expressed the highest pectinase productivity (0.76 U/mL) on 0.5% wheat bran-containing medium at 37°C for four days. A 58 kDa pectinase was purified from the four-day cultured medium fermented under optimized culture conditions with 7.24% of a recovery ratio and 0.51 U/mg of specific activity, respectively. The optimum temperature, optimum pH, thermal stability, and pH stability of the TKU050 pectinase were 50 °C, pH 6, <50 °C, and pH 6-9, respectively. The TKU050 pectinase was inhibited by sodium dodecyl sulfate and Cu2+. The reducing sugar obtained by hydrolyzing banana peel with TKU050 pectinase showed the growth-enhancing effect on the growth of four tested lactic acid bacteria.

Bioprocessing of Marine Chitinous Wastes for the Production of Bioactive Prodigiosin.

Recently, microbial prodigiosin (PG) has received much attention due to its numerous beneficial applications. The aim of this study was to establish the bioprocessing of marine chitinous wastes (MCWs) for the cost-effective preparation of PG. Of the MCWs, demineralized shrimp shell powders (de-SSP) were found to be a potential source of carbon/nitrogen (C/N) for PG production by bacterial fermentation using Serratia marcescens strains. Further, PG scale-up production was investigated in a 15 L bioreactor system, and the highest yield (6200 mg/L) was achieved during fermentation using 5 L of a novel-designed culture broth that included 1.60% C/N sources (a de-SSP/casein ratio of 7/3), 0.02% K2SO4, and 0.05% K2HPO4, with an initial pH of 6-7. Fermentation was conducted in the dark at 27.5 °C for 8.0 h. This study was the first to report on the utilization of shrimp wastes for cost-effective, large-scale (5 L/pilot) PG production with high productivity (6200 mg/L) in a short cultivation time. The combination of 0.02% K2SO4 and 0.05% K2HPO4 was also found to be a novel salt composition that significantly enhanced PG yield. The red compound was purified and confirmed as PG after analyzing its HPLC profile, mass, and UV/vis spectra. The purified PG was then tested for its bioactivities and showed effective anticancer activities, moderated antioxidant activities, and novel anti-NO effects.

Utilization of Seafood Processing By-Products for Production of Proteases by Paenibacillus sp. TKU052 and Their Application in Biopeptides' Preparation.

Microbial fermentation of by-products is a renewable and efficient technique in the development of a range of useful products. In this study, protease synthesis by Paenibacillus sp. TKU052 was carried out on culture media containing some common seafood processing by-products (SPBPs) as the sole source of carbon and nitrogen (C/N). The most suitable C/N nutrition source for the production of proteases was found to be 3.0% (w/v) demineralized crab shells powder (deCSP) and maximal enzyme activity of 4.41 ± 0.16 U/mL was detected on the third day of the culture. Two proteases (P1 and P2) with a similar molecular weight of 31 kDa were successfully isolated and purified from the 3-day deCSP-containing medium. Both P1 and P2 exhibited the highest activity of gelatin hydrolysis at pH 6 and 60 °C. The gelatin hydrolysates catalyzed by Paenibacillus TKU052 proteases were evaluated for biological activities, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, angiotensin-I converting enzyme (ACE) inhibition, and prebiotic activities. The gelatin hydrolysates expressed 31.76-43.95% DPPH radical scavenging activity and 31.58-36.84% ACE inhibitory activity, which was higher than those from gelatin. Gelatin hydrolysates also showed the growth-enhancing effect on Bifidobacterium bifidum BCRC 14615 with an increase to 135.70-147.81%. In short, Paenibacillus sp. TKU052 could be a potential strain to utilize crab shell wastes to produce proteases for bio-active peptides' preparation.