Screening of Novel Laccase Producers—Isolation and Characterization of Cold-Adapted Laccase from Kabatiella bupleuri G3 Capable of Synthetic Dye Decolorization

Psychrophilic laccases catalyzing the bond formation in mild, environmentally friendly conditions are one of the biocatalysts at the focus of green chemistry. Screening of 41 cold-adapted strains of yeast and yeast-like fungi revealed a new laccase-producing strain, which was identified as Kabatiella bupleuri G3 IBMiP according to the morphological characteristics and analysis of sequences of the D1/D2 regions of 26S rDNA domain and the ITS1–5,8S–ITS2 region. The extracellular activity of laccase in reaction with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at the optimal pH 3.5 was 215 U/L after 15 days of growth in a medium with waste material and 126 U/L after 25 days of cultivation in a defined medium. Copper (II) ions (0.4 mM), Tween 80 (1.0 mM) and ascorbic acid (5.0 mM) increased the production of laccase. The optimum temperature for enzyme operation is in the range of 30–40 °C and retains over 60% of the maximum activity at 10 °C. New laccase shows high thermolability—half-life at 40 °C was only 60 min. Enzyme degradation of synthetic dyes was the highest for crystal violet, i.e., 48.6% after 1-h reaction with ABTS as a mediator. Outcomes of this study present the K. bupleuri laccase as a potential psychrozyme for environmental and industrial applications.

Download Full-text

Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis

PeerJ ◽

10.7717/peerj.2679 ◽

2016 ◽

Vol 4 ◽

pp. e2679 ◽

Cited By ~ 7

Author(s):

Xi Bai ◽

Xianjun Yuan ◽

Aiyou Wen ◽

Junfeng Li ◽

Yunfeng Bai ◽

...

Keyword(s):

Symbiotic Bacterium ◽

Tween 80 ◽

Relative Activity ◽

Industrial Applications ◽

Biochemical Properties ◽

Recombinant Enzyme ◽

Enzymatic Properties ◽

Cold Adapted ◽

E Coli ◽

Thermal Asymmetric Interlaced Pcr

Background Many biotechnological and industrial applications can benefit from cold-adapted EglCs through increased efficiency of catalytic processes at low temperature. In our previous study, Citrobacter farmeri A1 which was isolated from a wood-inhabiting termite Reticulitermes labralis could secrete a cold-adapted EglC. However, its EglC was difficult to purify for enzymatic properties detection because of its low activity (0.8 U/ml). The objective of the present study was to clone and express the C. farmeri EglC gene in Escherichia coli to improve production level and determine the enzymatic properties of the recombinant enzyme. Methods The EglC gene was cloned from C. farmeri A1 by thermal asymmetric interlaced PCR. EglC was transformed into vector pET22b and functionally expressed in E. coli. The recombination protein EglC22b was purified for properties detection. Results SDS-PAGE revealed that the molecular mass of the recombinant endoglucanase was approximately 42 kDa. The activity of the E. coli pET22b-EglC crude extract was 9.5 U/ml. Additionally, it was active at pH 6.5–8.0 with an optimum pH of 7.0. The recombinant enzyme had an optimal temperature of 30–40 °C and exhibited >50% relative activity even at 5 °C, whereas it lost approximately 90% of its activity after incubation at 60 °C for 30 min. Its activity was enhanced by Co2+ and Fe3+, but inhibited by Cd2+, Zn2+, Li+, Triton X-100, DMSO, acetonitrile, Tween 80, SDS, and EDTA. Conclusion These biochemical properties indicate that the recombinant enzyme is a cold-adapted endoglucanase that can be used for various industrial applications.

Download Full-text

Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-β-1,4-xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433

Biomolecules ◽

10.3390/biom11050680 ◽

2021 ◽

Vol 11 (5) ◽

pp. 680

Author(s):

Do Young Kim ◽

Jonghoon Kim ◽

Yung Mi Lee ◽

Jong Suk Lee ◽

Dong-Ha Shin ◽

...

Keyword(s):

Tween 80 ◽

Maximum Activity ◽

Soil Bacterium ◽

Glycoside Hydrolase Family ◽

Antarctic Soil ◽

Birchwood Xylan ◽

Uncultured Bacterium ◽

Cold Adapted ◽

Beechwood Xylan ◽

Key Enzyme

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase.

Download Full-text

The Sustainable Production of a Novel Laccase from Wheat Bran by Bordetella sp. JWO16: Toward a Total Environment

Catalysts ◽

10.3390/catal11060677 ◽

2021 ◽

Vol 11 (6) ◽

pp. 677

Author(s):

John Onolame Unuofin

Keyword(s):

Wheat Bran ◽

Contaminated Soils ◽

Residual Activity ◽

Dye Decolorization ◽

Fermentation Medium ◽

Biochemical Properties ◽

Synthetic Dyes ◽

High Concentrations ◽

Capital Intensive ◽

Agroindustrial Residue

Laccase is increasingly adopted in diverse industrial and environmental applications, due to its readily accessible requirements for efficient catalytic synthesis and biotransformation of chemicals. However, it is perceived that its industrial production might incur some unfavorable overhead, which leads to expensive market products, and the corresponding negative environmental feedback, due to the use of capital-intensive and precarious chemicals. To this end, this study was designed to evaluate the performance indicators of the valorization of wheat bran by a novel Jb1b laccase and its subsequent application in waste minimization and water management, on a laboratory scale. Optimal Jb1b laccase was produced in submerged fermentation medium containing wheat bran, an agroindustrial residue, through response surface methodology (RSM) algorithm, and was applied in dye decolorization and denim bioscouring, respectively. Results showed that the resultant enzyme manifested unique biochemical properties, such as enhanced tolerance at certain physicochemical conditions, with a residual activity of at least ca. 76%. Furthermore, phenomenally high concentrations of synthetic dyes (0.2% w v−1) were decolorized over 56 h, and a 6 h mediator-supported simultaneous denim bleaching and decolorization of wash effluent was observed. The sustainability of the production and application processes were inferred from the reusability of the fermentation sludge as a potential biofertilizer, with subsequent prospects for the biostimulation and bioaugmentation of contaminated soils, whereas the decolorized water could be adopted for other uses, amongst which horticulture and forestry are typical examples. These phenomena therefore authenticate the favorable environmental feedbacks and overhead realized in this present study.

Download Full-text

Identification of bacteriological quality and antimicrobial resistance of microorganisms isolated from animal foods collected from the abattoir, butcher shops and local seafood market

Food Research ◽

10.26656/fr.2017.5(3).570 ◽

2021 ◽

Vol 5 (3) ◽

pp. 144-151

Author(s):

P. Vijayalakshmi ◽

A. Rajani Chowdary ◽

P. Vidyullatha ◽

M. Sharon Sushma

Keyword(s):

Antimicrobial Resistance ◽

Culture Media ◽

Morphological Characteristics ◽

Sampling Technique ◽

Antibiotic Use ◽

Multidrug Resistant ◽

Resistance Pattern ◽

Human Beings ◽

Isolation And Characterization ◽

Butcher Shops

The current study aimed to isolate bacteria that harbour various animal food products like meat, chicken and seafoods collected from the abattoir, butcher shops and local seafood market and to determine the antimicrobial resistance pattern of isolated pathogens which are responsible for various foodborne illnesses in human beings. A total of forty raw animal product samples were collected from the abattoir, butcher shops and local seafood market of Visakhapatnam. The samples selected for the study include raw chicken, meat, crab, prawns and different varieties of fish. A classic random sampling technique was employed to collect the study samples. All the samples were processed immediately using standard microbiological protocols. The bacteria isolation and characterization were done by studying morphological characteristics with staining methods, cultural characteristics by isolating and growing the pathogenic microorganisms in various selective and differential culture media. Antimicrobial susceptibility testing was performed by the Kirby -Bauer method by following Clinical and Laboratory Standards Institute (CLSI) guidelines. EDTA-Disc Potentiation Test and Imipenem-EDTA Double disc synergy test are used to detect the metallo beta-lactamase production of isolated pathogens. The highest number of isolates belong to Salmonella species (18), Pseudomonas aeruginosa (18) followed by Vibrio species (14) and few isolates belong to Enterobacter species (4). Majority of the microbial isolates obtained in the current study were multidrug resistant. The isolates from the abattoir environments, slaughterhouses, fish markets were found to exhibit variable resistance pattern to aminoglycosides, macrolides, β-lactams, cephalosporins, quinolone antibiotics used in the present study and at the same time most of them were sensitive to carbapenem antibiotic imipenem. Antimicrobial resistance (AMR) prevents the designing and assessment of effective interventions. If such a link can be established, then the tracking of antibiotic use and consumption data could be furthermore used as a surrogate indicator for the risk of potential antibiotic resistance (ABR) emergence.

Download Full-text

Isolation and characterization of bleomycin-resistant clones of CHO cells

Genetics Research ◽

10.1017/s0016672300019509 ◽

1979 ◽

Vol 34 (3) ◽

pp. 269-279 ◽

Cited By ~ 10

Author(s):

Sylvia Brabbs ◽

J. R. Warr

Keyword(s):

Enzyme Activity ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Tween 80 ◽

The Other ◽

Resistant Clone ◽

Ovary Cells ◽

Isolation And Characterization ◽

Significant Difference

SUMMARYFive clones of Chinese hamster ovary cells with increased resistance to bleomycin have been isolated following ethylmethanesulphonate mutagenesis. Resistance was stable in three of the clones, but unstable in the other two. One of the stably resistant clones was cross resistant to unrelated drugs, and in contrast to the parental cells, its response to bleomycin was potentiated by tween 80. These two observations suggested a membrane alteration in the resistant clone. There was no significant difference in bleomycin-inactivating enzyme activity between the parental and resistant clones.

Download Full-text

Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications

10.21203/rs.3.rs-19704/v1 ◽

2020 ◽

Author(s):

Wissal BEN ALI ◽

Delphine Chaduli ◽

David Navarro ◽

Christian Lechat ◽

Annick Turbé-Doan ◽

...

Keyword(s):

Culture Supernatant ◽

Cost Effective ◽

Optimal Growth ◽

Dye Decolorization ◽

Growth Conditions ◽

Active Cell ◽

Oxidative Enzymes ◽

Synthetic Dyes ◽

Biotechnological Applications ◽

Fungal Strains

Abstract Background : Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost–effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization.Results : Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum , Stemphylium lucomagnoense and Aspergillus nidulans . Among these five isolates, one T. asperellum strain ( T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator.Conclusions : The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO 4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

Download Full-text

First Report of Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum luteum Associated with Canker and Dieback of Grapevines in Tunisia

Plant Disease ◽

10.1094/pdis-05-13-0530-pdn ◽

2014 ◽

Vol 98 (3) ◽

pp. 420-420 ◽

Cited By ~ 6

Author(s):

S. Chebil ◽

R. Fersi ◽

A. Yakoub ◽

S. Chenenaoui ◽

M. Chattaoui ◽

...

Keyword(s):

Morphological Characteristics ◽

Aerial Mycelium ◽

Its2 Region ◽

Pathogenicity Test ◽

Botryosphaeria Dothidea ◽

Diplodia Seriata ◽

First Report ◽

Grapevine Dieback ◽

Pinus Spp ◽

Neofusicoccum Luteum

In 2011, common symptoms of grapevine dieback were frequently observed in 2- to 5-year-old table grape (Vitis vinifera L.) cvs. in four vineyards located in northern Tunisia. The symptoms included dead spur and cordons, shoot dieback, and sunken necrotic bark lesions, which progressed into the trunk resulting in the death of large sections of the vine. Longitudinal and transversal sections of cordons and spurs from symptomatic vines revealed brown wedge-shaped cankers of hard consistency. Twelve symptomatic samples from spur and cordons were collected, surface disinfected by dipping into 5% (v/v) sodium hypochlorite for 2 min, and small pieces from the edge of necrotic and healthy tissue were removed and plated onto potato dextrose agar (PDA) at 25°C in the dark. Based on colony and conidia morphological characteristics, isolates were divided in three species, named Diplodia seriata, Botryosphaeria dothidea, and Neofusicoccum luteum. D. seriata colonies were gray-brown with dense aerial mycelium producing brown cylindric to ellipsoid conidia rounded at both ends and averaged 22.4 × 11.7 μm (n = 50). B. dothidea colonies were initially white with abundant aerial mycelium, gradually becoming dark green olivaceous. Conidia were fusiform to fusiform elliptical with a subobtuse apex and averaged 24.8 × 4.7 μm (n = 50). N. luteum colonies were initially pale to colorless, gradually darkening with age and becoming gray to dark gray producing a yellow pigment that diffuses into the agar. Conidia were hyaline, thin-walled, aseptate, fusiform to fusiform elliptical, and averaged 19.8 × 5.5 μm (n = 50). Identity of the different taxa was confirmed by sequence analyses of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA and part of the elongation factor 1-alpha (EF1-α) gene. BLAST analysis of sequences indicated that six isolates were identified as D. seriata (GenBank: AY259094, AY343353), one isolate as B. dothidea (AY236949, AY786319) and one isolate as N. luteum (AY259091, AY573217). Sequences were deposited in GenBank under accessions from KC178817 to KC178824 and from KF546829 to KF546836 for ITS region and EF1-α gene, respectively. A pathogenicity test was conducted on detached green shoots cv. Italia for the eight Botryosphaeriaceae isolates. Shoots were inoculated by placing a colonized agar plug (5 mm diameter) from the margin of a 7-day-old colony on fresh wound sites made with a sterilized scalpel. Each wound was covered with moisturized cotton and sealed with Parafilm. Control shoots were inoculated using non-colonized PDA plugs. After 6 weeks, discoloration of xylem and phloem and necrosis with average length of 38.8, 17.6, and 11.2 mm were observed from inoculated shoots with D. seriata, N. luteum, and B. dothidea, respectively, and all three fungi were re-isolated from necrotic tissue, satisfying Koch's postulates. Control shoots showed no symptoms of the disease and no fungus was re-isolated. In Tunisia, Botryosphaeria-related dieback was reported only on citrus tree caused by B. ribis (2), on Pinus spp. caused by D. pinea (4), on Quercus spp. caused by D. corticola (3), and on olive tree (Olea europea) caused by D. seriata (1). To our knowledge, this is the first report of D. seriata, B. dothidea, and N. luteum associated with grapevine dieback in Tunisia. References: (1) M. Chattaoui et al. Plant Dis. 96:905, 2012. (2) H. S. Fawcett. Calif. Citrogr. 16:208, 1931. (3) B. T. Linaldeddu et al. J. Plant Pathol. 91:234. 2009. (4) B. T. Linaldeddu et al. Phytopathol. Mediterr. 47:258, 2008.

Download Full-text

Prospects of Biocatalyst Purification Enroute Fermentation Processes

10.5772/intechopen.97382 ◽

2021 ◽

Author(s):

Michael Bamitale Osho ◽

Sarafadeen Olateju Kareem

Keyword(s):

Fermentation Process ◽

Colloidal Particles ◽

Methyl Cellulose ◽

Industrial Applications ◽

Maximum Activity ◽

Inorganic Materials ◽

Organic Substrates ◽

Ammonium Sulphate Precipitation ◽

Sodom Apple ◽

Microbial Agents

Biotransformation of broth through fermentation process suffers a major setback when it comes to disintegration of organic substrates by microbial agents for industrial applications. These biocatalysts are in crude/dilute form hence needs to be purified to remove colloidal particles and enzymatic impurities thus enhancing maximum activity. Several contractual procedures of concentrating dilute enzymes and proteins had been reported. Such inorganic materials include ammonium sulphate precipitation; salting, synthetic polyacrylic acid; carboxy-methyl cellulose, tannic acid, edible gum and some organic solvents as precipitants etc. The emergence of organic absorbents such as sodom apple (Calostropis procera) extract, activated charcoal and imarsil had resulted in making significant impact in industrial circle. Various concentrations of these organic extracts have been used as purifying agents on different types of enzyme vis: lipase, amylase, protease, cellulase etc. Purification fold and stability of the enzyme crude form attained unprecedented results.

Download Full-text

A novel Swollenin from Talaromyces leycettanus JCM12802 exhibits cellulase activity and enhanced cellulase hydrolysis towards various substrates

10.21203/rs.3.rs-22793/v1 ◽

2020 ◽

Author(s):

Honghai Zhang ◽

Yuan Wang ◽

Roman Brunecky ◽

Bin Yao ◽

Xiangming Xie ◽

...

Keyword(s):

Synergistic Effect ◽

Functional Diversity ◽

Specific Activity ◽

Biomass Conversion ◽

Hydrolytic Enzymes ◽

Cellulase Activity ◽

Industrial Applications ◽

Maximum Activity ◽

Carbohydrate Binding ◽

Cell Wall Polysaccharides

Abstract Background Swollenins are present in some fungal species involved in the biodegradation of cellulosic substrates. They appear to promote a rearrangement in the network of non-covalent interactions between the cell wall polysaccharides, thus making it more accessible for degradation by hydrolytic enzymes. Here, we have reported a detailed characterization of a recombinant swollenin with respect to its disruptive activity on cellulosic substrates and synergistic effect with cellulases. Results In the present study, a novel swollenin gene Tlswo consisting of an open reading frame encoding 503 amino acids was identified from Talaromyces leycettanus JCM12802 and successfully expressed in Trichoderma reesei and Pichia pastoris. Similar to other fungal swollenins, TlSWO contained a N-terminal family 1 carbohydrate binding module (CBM1) followed by a Ser/Thr rich linker connected to expansin-like domain which includes a family 45 endoglucanase-like domain and group-2 grass pollen allergen domain. TlSWO demonstrated disruptive activity on Avicel and displayed a high synergistic effect with cellobiohydrolases, enhancing its hydrolytic performance up to 132%. The activity of TlSWO on various substrates and biomass was also examined. It was shown that TlSWO could release reducing sugars from lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin. The specific activity of TlSWO towards the substates above is 9.0 ± 0.100 U/mg, 8.9 ± 0.100U/mg, 2.3 ± 0.002 U/mg and 0.79 ± 0.002 U/mg respectively. Moreover, TlSWO exhibits maximum activity at pH 4.0 and 50 ℃. Conclusion This study reported on a novel swollenin with highly efficient for biomass conversion. It also reveals the functional diversity of swollenin with activity on various substrates. Although the exact mechanism of swollenin catalytic action activity still remains unknown, the functional diversity of TlSWO makes it a good candidate for industrial applications.

Download Full-text