scholarly journals OPTIMIZING THE PRODUCTION OF A FUNCTIONAL TYPE A RECOMBINANT ENDOCHITINASE FROM Trichoderma asperellum IN Escherichia coli

2021 ◽  
Vol 9 (6) ◽  
pp. 871-880
Author(s):  
Nguyen Ngoc Luong ◽  
Nguyen Quang Duc Tien ◽  
Phung Thi Bich Hoa ◽  
Nguyen Hoang Tue ◽  
Mai Thi Thu Hien ◽  
...  

Chitinases from the genus Trichoderma fungi are mainly responsible for their anti-fungal activities, which allow them to become the most widely used fungal biocontrol. Therefore, several Trichoderma chitinases have been cloned and expressed to facilitate their production and applications. A previous study of the same authors has characterized an endochitinase from a relatively novel Trichoderma spp., Trichoderma asperellum. To produce this enzyme more economically and efficiently, we reported the synthesis and expression of its synthetic encoding gene in the Escherichia coli M15 strain and established the optimal conditions for preparative scale production of the enzyme in its functional form. By lowering the induction temperatures, we observed substantial improvement in the expression levels of the active enzyme.  At 30 oC and 0.5 mM IPTG induction, 1 L of cells yielded approximately 80 - 100 mg of soluble protein, accounting for about 9-11 % of total soluble protein. This figure may be an underestimation of the actual yield, as deduced from the SDS-PAGE data. The recombinant enzyme can be retrieved by simple repeated freezing and thawing cycles and purified to near homogeneity using Ni-NTA chromatography. The purified enzyme showed in vitro colloidal chitin hydrolysis activity. These results could be scaled up to produce soluble 42 kDa chitinase in E. coli. The study demonstrated an economical method to produce chitinases for various agricultural and environmental applications.

1994 ◽  
Vol 72 (1) ◽  
pp. 188-192 ◽  
Author(s):  
Kazuki Saito ◽  
Reiko Kanda ◽  
Makoto Kurosawa ◽  
Isamu Murakoshi

Cysteine synthase (EC 4.2.99.8) in higher plants is responsible for biosynthesis of not only cysteine but also some nonprotein amino acids such as β-(pyrazol-1-yl)-L-alanine. The cDNA of a cysteine synthase from spinach (Spinacia oleracea) was inserted into pET8c (=pET3d) under the transcriptional control of strong T7 promoter to yield an overexpression vector pCEK1. The amount of the exogenous cysteine synthase was increased up to 40% of the total soluble protein of Escherichia coli transformed with pCEK1. β-(Pyrazol-1-yl)-L-alanine, a specific metabolite in plants of the Cucurbitaceae, was biosynthesized by overexpressed cysteine synthase from pyrazole in the presence of O-acetyl-L-serine and serine, in vitro and in vivo, respectively. The present study provides the system for mechanistic investigation of biosynthesis of cysteine and biogenetically related β-substituted alanines at molecular genetic level.


2020 ◽  
Vol 21 (24) ◽  
pp. 9640
Author(s):  
Ellen Y. Cotrina ◽  
Marta Vilà ◽  
Joan Nieto ◽  
Gemma Arsequell ◽  
Antoni Planas

Human transthyretin (hTTR), a serum protein with a main role in transporting thyroid hormones and retinol through binding to the retinol-binding protein, is an amyloidogenic protein involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis. hTTR also has a neuroprotective role in Alzheimer disease, being the major Aβ binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-β (Aβ) aggregation with consequent abrogation of toxicity. Here we report an optimized preparative expression and purification protocol of hTTR (wt and amyloidogenic mutants) for in vitro screening assays of TTR ligands acting as amyloidogenesis inhibitors or acting as molecular chaperones to enhance the TTR:Aβ interaction. Preparative yields were up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. The recombinant wt protein is mainly unmodified at Cys10, the single cysteine in the protein sequence, whereas the highly amyloidogenic Y78F variant renders mainly the S-glutathionated form, which has essentially the same amyloidogenic behavior than the reduced protein with free Cys10. The TTR production protocol has shown inter-batch reproducibility of expression and protein quality for in vitro screening assays.


2011 ◽  
Vol 345 ◽  
pp. 134-138 ◽  
Author(s):  
Li Hui Lv ◽  
Xue Gang Luo ◽  
Meng Ni ◽  
Xiao Lan Jing ◽  
Nan Wang ◽  
...  

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.


1984 ◽  
Vol 219 (1) ◽  
pp. 277-285 ◽  
Author(s):  
J C Díez ◽  
M Little ◽  
J Avila

Tubulin from pig lung was quantitatively determined, isolated and characterized. It accounted for about 0.3-0.4% of the total soluble protein of pig lung, as measured by colchicine binding or radioimmunoassay. Purified tubulin was obtained by several cycles of polymerization and depolymerization in the presence of dimethyl sulphoxide and 2H2O as stabilizing agents. The proteolytic cleavage patterns of the lung tubulin subunits closely resembled those of other mammalian cytoplasmic tubulin subunits, such as those of brain and kidney. However, the pattern of lung isotubulins on isoelectric focusing differed substantially from that of brain isotubulins . These differences did not appear to be the result of major lung tubulin post-translational modifications, since approximately the same pattern of isotubulins was found for the tubulin synthesized by lung poly(A)-containing mRNA in a reticulocyte system in vitro.


2021 ◽  
Author(s):  
Kamila Lourrane Carvalho Alencar Rocha ◽  
Kárita Cristine Rodrigues Dos Santos ◽  
Maraiza Castro Bezerra ◽  
Vanice Conceição Do Nascimento

Introdução: O fitopatógeno Sclerotinia sclerotiorum, causador da doença conhecida popularmente como mofo-branco, acomete diversas culturas agrícolas em todo o mundo, sendo a soja uma das principais monoculturas mais afetadas. Seu alto grau de patogenicidade está associado à sua capacidade de formar escleródios que atuam como mecanismo de resistência e sobrevivência, podendo permanecer viáveis no solo por mais de dez anos e, em muitos casos, são resistentes aos agroquímicos. Considerando a necessidade de métodos biotecnológicos que contribuam no desenvolvimento agrícola sem promover danos ao ambiente e a população, os agentes de controle biológico (ACBs) são hoje vistos como um excelente artifício. Fungos do gênero Trichoderma podem ser considerados ACBs sagazes por apresentar inúmeros mecanismos com alto potencial antagônico a diversos fitopatógenos, que prejudicam a germinação de esporos, o crescimento das hifas e o desenvolvimento de escleródios. Objetivo: Avaliar o potencial antagônico de isolados de Trichoderma em inibir o crescimento micelial do fitopatógeno S. sclerotiorum através da liberação de metabólitos voláteis. Material e Métodos: Para análise da produção de metabólitos voláteis, os isolados Trichoderma harzianum ALL-42 e o Trichoderma asperellum TR-356 e o fitopatógeno S. sclerotiorum, foram dispostos em fundos de placas de Petri com meio Batata Dextrose Ágar (BDA), em face opostas ao outro, onde, no inferior estava o isolado de Trichoderma e no superior o fitopatógeno. Uma placa controle contendo apenas o fitopatógeno foi feita para comparação dos resultados. Resultados: Ambos isolados de Trichoderma apresentaram resultados positivos na inibição do crescimento micelial de S. sclerotiorum. Conclusão: A produção de metabólitos voláteis é uma ação secundária do gênero Trichoderma, ou seja, sua sobrevivência não depende desse mecanismo, ainda assim, esses metabólitos agem contra os antagonistas presentes no meio. Com isso, podemos verificar o quão eficiente é esse gênero como agente de controle biológico, entretanto, outros isolados precisam ser testados no intuito de encontrarmos agentes mais especialistas na defesa contra o fitopatógeno S. sclerotiorum, e dessa forma instigar os benefícios do emprego de agentes biotecnológicos no campo, visando a redução da aplicação de produtos químicos na agricultura.


Weed Science ◽  
1980 ◽  
Vol 28 (4) ◽  
pp. 393-400 ◽  
Author(s):  
Robert E. Hoagland

The growth regulator, glyphosine [N,N-bis(phosphonomethyl)glycine], and other possible metabolites of glyphosine and glyphosate [N-(phosphonomethyl)glycine] [glycine, sarcosine, and aminomethylphosphonic acid (AMPA)] were tested individually (0.5 mM) or as a mixture (each at 0.5 mM) for their effects on growth, extractable phenylalanine ammonia-lyase (PAL) activity, hydroxyphenolic-compound production, chlorophyll and anthocyanin contents, and on soluble-protein levels in soybean [Glycine max(L.) Merr. ‘Hill’] seedlings. Most chemical treatments caused some inhibition of growth either on fresh weight accumulation or on root elongation in the light and dark over 72 h. Glyphosine was generally the most inhibitory and caused the greatest inhibition on axis dry-weight accumulation. Glyphosine significantly increased extractable PAL activity in axes of light- and dark-grown soybeans to a lesser extent than did glyphosate. AMPA had some inhibitory effects on extractable PAL activity whereas other compounds had little influence on the enzyme. These compounds had little effect on total soluble protein in axes or on soluble protein in PAL preparations from 12 to 72 h in light-or dark-grown seedlings. No in vitro effect of the chemicals on PAL activity was found at concentrations up to 0.5 mM. Hydroxyphenolic compound levels increased within 24 to 72 h (per gram fresh weight basis) in light- or dark-grown soybean axes treated with glyphosine, AMPA, or a metabolite mixture (AMPA, sarcosine, and glycine). Anthocyanin content was decreased by glyphosate and to a lesser extent by glyphosine, but was increased by AMPA and the mixture. Glyphosate significantly increased the chlorophylla/bratio and decreased total chlorophyll, but glyphosine decreased the chlorophyll content to a lesser degree.


2021 ◽  
Vol 17 (6) ◽  
pp. 805-817
Author(s):  
Sharifah Zafierah Syed Badrulzaman ◽  
Nurhana Nadia Ramlan ◽  
Munirah Adibah Kamarul Zaman ◽  
Azzreena Mohamad Azzeme

Musa paradisiaca cv Lang belongs to cooking banana group, and it has high potential to be used in banana chips production. Like other cultivars, M. paradisiaca cv Lang is susceptible towards water shortage, therefore affecting banana growth and productivity. In this study, to mimic the drought condition, pseudo-drought stress was given to in vitro Lang banana seedlings by adding polyethylene glycol (PEG). Overall, decrement of roots length and chlorophyll (Chl) content was displayed by the seedlings exposed to 1%, 2%, 3%, 4%, and 5% (w/v) of PEG after three weeks of exposure. The proline content, total soluble protein content, and antioxidant capacity in leaf and roots, however, countered differently towards different levels of drought. Proline content showed the highest in leaf of 2% (w/v) PEG-treated seedling (12.66±0.38 µmoles/g) while the total soluble protein content showed the highest in roots of 5% (w/v) of PEG-treated seedling (30.65±1.07 mg/g FW). Antioxidant capacity of stressed seedlings revealed the catalase (CAT), guaiacol peroxidase (POD), and ascorbate peroxidase (APX) activities were the highest in the leaf of 1% (w/v) (10.69±5.06 µmol/min/mg), 4% (w/v), (0.079±0.03 µmol/min/mg), and 5% (w/v) (9.11±8.47 µmol/min/mg) of PEG- treated seedlings, respectively. Meanwhile, the highest CAT, POD, and APX activities in the roots were determined in 3% (w/v) (0.49±0.04 µmol/min/mg), 2% (w/v) (0.03±0.02 µmol/min/mg), and 3% (w/v) (16.69±0.5 µmol/min/mg) of PEG-treated seedlings, respectively. These data show that PEG can be a priming agent to induce defense system at seedling stage of banana, which could enhance their survivability during ex vitro acclimatization.


Author(s):  
Ryotaro Hara ◽  
Yuta Nakajima ◽  
Hiroaki Yanagawa ◽  
Ryo Gawasawa ◽  
Izumi Hirasawa ◽  
...  

β-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of β-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo -β-selective hydroxylation of l -His and l -Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l - threo -β-hydroxy-His and l - threo -β-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g L −1 ) l - threo -β-hydroxy-His and 150 mM l - threo -β-hydroxy-Gln (24.3 g L −1 ) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l -His/ l -Gln threo -β-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of β-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. Importance Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for β-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l -His and l -Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l - threo -β-hydroxy-His and l - threo -β-hydroxy-Gln with a titer of over 20 g L −1 through whole-cell bioconversion using recombinant Escherichia coli cells. As β-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.


2009 ◽  
Vol 21 (3) ◽  
pp. 175-186 ◽  
Author(s):  
Jamile F. Gonçalves ◽  
Alexssandro G. Becker ◽  
Luciane B. Pereira ◽  
João B. T. da Rocha ◽  
Denise Cargnelutti ◽  
...  

In this study, the effects of lead (Pb) on growth, photosynthetic pigments concentration, lipid peroxidation, electrolyte leakage percentage (ELP), protein oxidation, aminolevulinate dehydratase (ALA-D; E.C. 4.2.1.24), ascorbate peroxidase (APX; E.C. 1.11.1.11), catalase (CAT; E.C. 1.11.1.6) and superoxide dismutase (SOD; E.C. 1.15.1.1) activities, and ascorbic acid (AsA), non-protein thiol groups (NPSH) and total soluble protein concentrations in cucumber seedlings (Cucumis sativus L.) were investigated. Seedlings were grown in vitro in an agar-solidified substrate containing three Pb levels as (C2H3O2)Pb.3H2O (0, 100, 400, and 1000 µmol L-1) for 10 d. Increasing Pb concentrations in substrate enhanced Pb concentration in both roots and shoot. Pb accumulated at a higher amount in roots. Root length and total fresh weight were decreased at the two highest Pb concentrations. Cucumber showed no reduction in shoot length and total dry weight at any Pb level. The highest Pb concentration decreased water content and ALA-D activity as well as increased malondialdehyde, carbonyls and total soluble protein concentrations. Carotenoids concentration enhanced at 100 and 400 µmol Pb L-1, while chlorophyll concentration and ELP were not affected by Pb stress. Activity of APX was inhibited while the activities of CAT and SOD were increased at all Pb concentrations. AsA was enhanced at 400 and 1000 µmol Pb L-1 whereas NPSH were increased only at the highest Pb concentration. Therefore, high Pb-exposure caused oxidative stress, and the antioxidant system of the cucumber seedlings was not sufficient to revert it, contributing for growth reduction.


Plant Disease ◽  
2019 ◽  
Vol 103 (11) ◽  
pp. 2733-2741 ◽  
Author(s):  
Hao Chou ◽  
Yi-Ting Xiao ◽  
Jyh-Nong Tsai ◽  
Ting-Ting Li ◽  
Hung-Yi Wu ◽  
...  

Brown root rot (BRR), caused by the white rot fungus Phellinus noxius, is an epidemic disease of diverse broadleaved and coniferous tree species in many tropical and subtropical regions. Flooding and trenching control measures are difficult to implement, and chemical controls can have an adverse impact on ecosystems. Previous studies have provided in vitro evidence for the potential use of Trichoderma spp. for biocontrol of BRR. Here, we analyzed the in vitro antagonistic and mycoparasitic abilities of four Trichoderma spp. isolates against four P. noxius isolates in dual culture and Ficus microcarpa wood blocks. A convenient inoculation system based on root inoculation of a highly susceptible loquat (Eriobotrya japonica) with P. noxius-colonized wheat-oat grains was developed to examine the effect of Trichoderma treatment in planta. Preventive application of Trichoderma asperellum TA, the isolate showing high antagonistic activity in vitro, was effective in preventing and delaying the wilting of P. noxius-inoculated loquat cuttings in greenhouse trials. To understand the specific niche in which T. asperellum TA interacts with P. noxius, KOH-aniline blue fluorescence microscopy was used to investigate the colonization of loquat roots by P. noxius and/or T. asperellum TA. Dilution plating assays were also conducted to quantify Trichoderma populations in the rhizosphere and potting mix. T. asperellum TA was able to robustly establish in the rhizosphere and potting mix but with scarce root penetration limited to the superficial layer. We discuss the timing and strategy for applying antagonistic Trichodema sp. on living trees or in BRR-infested areas for BRR management.


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