Molecular Modification Research Articles

Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.

1,5,6,7-Tetrahydro-4H-indazol-4-ones as human neutrophil elastase (HNE) inhibitors

Human neutrophil elastase (HNE) is a serine protease that is expressed in polymorphonuclear neutrophils. It has been recognized as an important therapeutic target for treating inflammatory diseases, especially related to the respiratory system, but also for various types of cancer. Thus, compounds able to inhibit HNE are of great interest in medicinal chemistry. In the present paper, we report the synthesis and biological evaluation of a new series of HNE inhibitors with an innovative 1,5,6,7-tetrahydro-4H-indazol-4-one core that was developed as a molecular modification of our previously reported indazole-based HNE inhibitors. Since the 1,5,6,7-tetrahydro-4H-indazol-4-one scaffold can occur in two possible tautomeric forms, the acylation/alkylation reactions resulted in a mixture of the two isomers, often widely unbalanced in favor of one form. Using analytical techniques and NMR spectroscopy, we characterized and separated the isomer pairs and confirmed the compounds used in biological testing. Analysis of the compounds for HNE inhibitory activity showed that they were potent inhibitors, with Ki values in the low nanomolar range (6–35 nM). They also had reasonable stability in aqueous buffer, with half-lives over 1 h. Overall, our results indicate that the 1,5,6,7-tetrahydro-4H-indazol-4-one core is suitable for the synthesis of potent HNE inhibitors that could be useful in the development of new therapeutics for treating diseases involving excessive HNE activity.

Efficient Production of Scleroglucan by Sclerotium rolfsii and Insights Into Molecular Weight Modification by High-Pressure Homogenization.

Scleroglucan is a non-ionic water-soluble polysaccharide, and has been widely used in the petroleum, food, medicine and cosmetics industries. Currently, scleroglucan is mainly produced by Sclerotium rolfsii. A higher level of scleroglucan (42.0 g/L) was previously obtained with S. rolfsii WSH-G01. However, the production of scleroglucan was reduced despite a higher glucose concentration remaining. Additionally, the molecular weight of scleroglucan was large, thus restricted its application. In this study, by adjusting the state of seeds inoculated, the degradation issue of scleroglucan during the fermentation process was solved. By comparing different fed-batch strategies, 66.6 g/L of scleroglucan was harvested by a two-dose fed-batch mode, with 53.3% glucose conversion ratio. To modify the molecular weight of scleroglucan, a combination method with HCl and high-pressure homogenization treatment was established. Finally, scleroglucan with molecular weight of 4.61 × 105 Da was obtained. The developed approaches provide references for the biosynthesis and molecular weight modification of polysaccharides.

Molecular Manipulation of the MiR396/GRF Expression Module Alters the Salt Stress Response of Arabidopsis thaliana

We previously demonstrated that microRNA396 (miR396) abundance is altered in 15-day-old Arabidopsis thaliana (Arabidopsis) whole seedlings following their exposure to a 7-day salt stress treatment regime. We, therefore, used a molecular modification approach to generate two new Arabidopsis transformant populations with reduced (MIM396 plants) and elevated (MIR396 plants) miR396 abundance. The exposure of 8-day-old wild-type Arabidopsis whole seedlings and a representative plant line of the MIM396 and MIR396 transformant populations to a 7-day salt stress treatment regime revealed unique phenotypic and physiological responses to the imposed stress by unmodified wild-type Arabidopsis plants and the MIM396 and MIR396 transformat lines. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was, therefore, applied to demonstrate that the plant line specific responses to salt stress likely stemmed from the unique molecular profile of each of the GROWTH REGULATING FACTOR (GRF) transcription factor gene family members which form posttranscriptional targets of miR396-directed expression regulation. RT-qPCR additionally revealed that, in 15-day-old Arabidopsis whole seedlings, the three previously identified putative target genes of miR396 belonging to the NEUTRAL/ALKALINE NONLYSOSOMAL CERAMIDASE-LIKE (NCER) gene family, including NCER1, NCER2, and NCER3, do not form targets of miR396-directed expression regulation at the posttranscriptional level. Taken together, the phenotypic and molecular analyses presented here demonstrate that alteration of the miR396/GRF expression module is central to the molecular response of Arabidopsis to salt stress.

Method for controlled tissue theranostics using a single tunable laser source.

Tissue diseases and related disorders need to be first recognized using diagnostic methods and then later treated by therapeutic methods-a joint procedure called theranostics. One of the main challenges in the field of retinal therapies remains in the success of the treatment, typically improving the local metabolism, by sparing the surrounding tissue and with the immediate information of the laser effect. In our study, we present a concept for real-time controlled tissue theranostics on a proof-of-concept study capable of using a single tunable ps laser source (in terms of irradiance, fluence, and repetition rate), done on ex-vivo human retinal pigment epithelium. We have found autofluorescence intensity and lifetime imaging diagnostics very promising for the recognition and quantification of laser effects ranging from selective non-destructive molecular tissue modification to complete tissue ablation. The main novelty of our work presents the developed algorithm for optimized theranostics based on the model function used to quantify laser-induced tissue changes through the diagnostics descriptors, fluorescence lifetime and fluorescence intensity parameters. This approach, together with the operation of the single adaptable laser source, can serve as a new theranostics method in personalized medicine in the future not only limited to treat retinal diseases.

Recent Progress of Extracellular Vesicle Engineering.

Extracellular vesicles (EVs) are nanoscale phospholipid bilayer membrane vesicles which contain varied active biomolecules. As natural carriers, EVs can deliver endogenous cargos to target tissues safely and effectively. However, the applications of natural released EVs are limited by their low yield and heterogeneity. Engineering EVs can endow them with more functions and better performances to address these issues. EVs can be modified and engineered to improve the yield, targeting efficiency, and content of beneficial cargos. Herein, the strategies of engineering EVs through genetic modification of EVs are introduced; the molecular modification of the EV membrane and the loading of nucleic acids are summarized; the building of EV mimetic nanovesicles are reviewed. Overall, we anticipate that readers will gain a better understanding of the progress of EV engineering, which will help to promote the development of the technologies and applications in this field.

Molecular Modification: A Tool for Pruning the Molecule

Modern Approaches in Drug Designing Molecular Modification: A Tool for Pruning the Molecule John Sunil R1*, Sridhar V1, Sarbani Pal2 and Jayashree A2 1Department of Pharmaceutical Chemistry, India 2Department of Chemistry, India *Corresponding author: John Sunil R, Department of Pharmaceutical Chemistry, Karimnagar, Telangana, India Submission: July 30, 2021;Published: August 26, 2021 DOI: 10.31031/MADD.2021.03.000565 ISSN: 2576-9170 Volume3 Issue3

Effect of lipoxygenase‐induced oxidation on molecular structure and digestive properties of arachin and conarachin

Lipid oxidation products can affect the molecular structure and digestibility of peanut protein isolates. Lipid-oxidizing systems (LOS) is a simulation system which uses oxygen-free radicals generated by the reaction of linoleic acid and lipoxygenase as oxidants.The simulation system was established to mediate oxidation of arachin or conarachin. Degree of oxidation (DOX) of peanut protein isolates (PPIs) was measured as quantity of LA to generate LOS. This study analyzed the effect of lipid oxidation-derived free radicals on the molecular structure and digestive properties of arachin and conarachin and observed substantial changes in carbonyl content, total sulfhydryl content, and number of disulfide bonds as quantities of LA increased, thus indicating significant oxidation of two proteins. Further, it was found that arachin was more stable than conarachin in a LOX-catalyzed oxidizing system. When the amount of LA was 3 and 5 ml, the maximum surface hydrophobic characteristic values of arachin and conarachin reached the maximum, which were 235 ± 10 and 349 ± 13, respectively. This study laid a theoretical foundation for the application of peanut protein in food products. Practical applications peanut protein has generated interest because it is the best source for the wall material for the preparation of nanoparticles and microcapsule particles due to its good biocompatibility and biodegradability, The structure-activity relationship between arachin and conarachin is of great guiding significance for the improvement of peanut protein molecular modification and functional properties.This study laid a theoretical foundation for the application of peanut protein in food products.

Research status of Bacillus phytase.

Phytic acid is abundant in seeds, roots and stems of plants, it acts as an anti-nutrient in food and feed industry, since it affects the absorption of nutrients by humans and monogastric animals. Furthermore, phosphorus produced through its decomposition by microorganisms can cause environmental pollution. Phytase degrades phytic acid generating precursors of inositol that can be used in clinical practice; in addition, phytase treatment can minimize the anti-nutritional effect of phytic acid. The use of phytase synthesized from Bacillus is more advantageous due to its high activity. Additionally, its good heat resistance under neutral conditions greatly fills the gap of commercial utilization of acid phytase. In this review, we summarize the latest research results on Bacillus phytase, including its physiological and biochemical characteristics, molecular structure information, calcium effects on its catalytic activity and stability, its catalytic mechanism and molecular modification.

Delineation of molecular structure modification during coagulation of mixed camel and cow milk by mid‐infrared spectroscopy and parallel factor analysis

Molecular structure modifications of camel milk (CaM) and cow milk (CM) mixtures during coagulation were investigated combining mid-infrared (MIR) spectroscopic monitoring with Parallel Factor Analysis (PARAFAC) and particle-size measurements. To evaluate the structure evolution at the molecular level, five different milk formulations were prepared using the following volume fractions of CaM in the mixtures: 100, 75, 50, 25, and 0. Regarding MIR spectroscopy, wavelength ranges located between 3,000 and 2,800 cm−1 corresponding to fatty acids; from 1,700 to 1,500 cm−1, related to amide I and II bands; and in the 1,500–900 cm−1 range called the fingerprint region were considered for the characterization of milk coagulation kinetics. MIR spectroscopy with PARAFAC was allowed for the identification of modifications at the molecular level depending on the coagulation time and milk composition. This was also confirmed by canonical correlation analysis, which demonstrated a high degree of correlation between the casein particle-size distributions and MIR spectra measured during coagulation. Novelty impact statement PARAFAC coupled with MIR spectroscopy were successfully used to monitor coagulation of CM, CaM, and their mixtures (CaM, CM, 1CaM:1CM, 1CaM:3CM, 3CaM:CM1; v/v). PARAFAC coupled with MIR spectroscopy were successfully used to differentiate between physicochemical phenomenon occurring during coagulation of different mixtures of CM, CaM and their mixtures. Canonical correlation analysis (CCA) of MIR spectra and particle-size distribution during milk coagulation demonstrated a strong relationship between the formation of casein aggregates and the variation of the MIR spectral data.

Design and Synthesis In Silico Drug-like Prediction and Pharmacological Evaluation of Cyclopolymethylenic Homologous of LASSBio-1514.

Acylhydrazones are still an important framework to the design of new bioactive compounds. As treatment of chronic pain represents a clinical challenge, we decided to modify the structure of LASSBio-1514 (1), previously described as anti-inflammatory and analgesic prototype. Applying the homologation as a strategy for molecular modification, we designed a series of cyclopentyl- (2a–e), cyclobutyl- (3a–e), and cyclopropylacylhydrazones (4a–e) that were synthetized and evaluated in murine models of inflammation and pain. A comparison of their in silico physicochemical and drug-like profile was conducted, as well as their anti-inflammatory and analgesic effect. Compounds 4a (LASSBio-1755) and 4e (LASSBio-1757) displayed excellent in silico drug-like profiles and were identified as new analgesic lead-candidates in acute and chronic model of pain, through oral administration.

Lipids and Lipoproteins in Health and Disease: Focus on Targeting Atherosclerosis.

Despite advances in pharmacotherapy, intervention devices and techniques, residual cardiovascular risks still cause a large burden on public health. Whilst most guidelines encourage achieving target levels of specific lipids and lipoproteins to reduce these risks, increasing evidence has shown that molecular modification of these lipoproteins also has a critical impact on their atherogenicity. Modification of low-density lipoprotein (LDL) by oxidation, glycation, peroxidation, apolipoprotein C-III adhesion, and the small dense subtype largely augment its atherogenicity. Post-translational modification by oxidation, carbamylation, glycation, and imbalance of molecular components can reduce the capacity of high-density lipoprotein (HDL) for reverse cholesterol transport. Elevated levels of triglycerides (TGs), apolipoprotein C-III and lipoprotein(a), and a decreased level of apolipoprotein A-I are closely associated with atherosclerotic cardiovascular disease. Pharmacotherapies aimed at reducing TGs, lipoprotein(a), and apolipoprotein C-III, and enhancing apolipoprotein A-1 are undergoing trials, and promising preliminary results have been reported. In this review, we aim to update the evidence on modifications of major lipid and lipoprotein components, including LDL, HDL, TG, apolipoprotein, and lipoprotein(a). We also discuss examples of translating findings from basic research to potential therapeutic targets for drug development.

The coordinated tunning optical, electrical and thermal properties of spiro-configured phenyl acridophopsphine oxide and sulfide for host materials

Two spiro-configured phenyl acridophopsphine-based oxide and sulfide host materials, named 5-phenyl-5H-spiro[acridophosphine- 10,9′-fluorene]-5-oxide (PSAFO) and 5-phenyl-5H-spiro [acridopho-sphine-10,9′-fluorene]5-sulfide (PSAFS), were synthesized by convenient method. The lowest unoccupied molecular orbital (LUMO) energy level and electron mobility could be decreased and improved as the S-atom replaced O-atom, respectively. Then, the electron injecting/transporting ability of PSAFS is expected to be enhanced. Moreover, the S-atom would provide PSAFS with better electron-donating capability and heavier molecular weight compared with that of PSAFO. Thus, a higher HOMO (the highest occupied molecular orbital) energy level and thermal stability can be achieved due to the shallower onset of oxidation potential and increased molecular weight. Consequently, a narrower energy gap between HOMO and LUMO of PSAFS would be obtained, and its thermal stability could be improved, which is beneficial to constructing high performance device. Although the frontier molecular orbitals and thermal stability were finely controlled by molecular modification, the high triplet (T 1 ) energy level was still retained due to the limited π-conjugation. With the high T 1 , HOMO and LUMO energy levels of 2.83/2.82, −6.19/-5.66 and −2.12/-2.19 eV, PSAFO and PSAFS based blue TADF OLEDs with low driving voltages of 2.9/3.1 V and maximum EQEs of 12.4/13.8% are successfully realized. • The first spiro-configured phenyl acridophopsphine-based oxidized compounds as hosts for TADF OLEDs. • A facile method was employed to tune the optical, electrical and thermal properties of the hosts collaboratively. • The spiro-configured phenyl acridophopsphine-based oxidized compounds could successfully suppress the concentration quenching of the guest demonstrated by the blue TADF OLEDs with high performance at the high doping ratio of 30%.

Molecular structural modification of egg white protein by pH-shifting for improving emulsifying capacity and stability

The effect of pH-shifting on the molecular structure and emulsifying properties of egg white protein (EWP) was investigated. EWP was subjected to extreme alkaline pH treatment (11, 11.5, 12, 12.5, and 13) and then adjusted to neutral (pH 7.0). The results showed that the emulsifying activity and emulsion stability of EWP were enhanced under pH-shifting, especially at pH 13. The surface hydrophobicity of EWP13 increased significantly from 306.94 to 11653.67 (p < 0.05), and the free sulfhydryl content of EWP13 increased significantly from 15.106 μmol/g to 121.86 μmol/g (p < 0.05). Also, the size and turbidity of EWP13 were minimal, producing EWP soluble aggregates with an average size of 52.90 nm (p < 0.05). Moreover, circular dichroism (CD) and intrinsic fluorescence spectra showed that the secondary and tertiary structures changed. At EWP12.5 and EWP13, it showed a decrease of α-helix and an increase of β-sheet. The intrinsic fluorescence spectra suggested that Phe, Tyr, and Trp were exposed to a polar environment due to EWP unfolding. In conclusion, the EWP structure was changed by pH-shifting, resulting in more exposed hydrophobic groups, thus increasing the emulsion stability. Correspondingly, the emulsion of EWP13 showed the best stability under the conditions of oxidation, heating, and salt than native EWP. This work may provide an effective method and theoretical basis for molecular modification to improve the functional properties of EWP by pH-shifting treatment.

Morphology Transformation of Foldamer Assemblies Triggered by Single Oxygen Atom on Critical Residue Switch.

The synthesis of morphologically well-defined peptidic materials via self-assembly is challenging but demanding for biocompatible functional materials. Moreover, switching morphology from a given shape to other predictable forms by molecular modification of the identical building block is an even more complicated subject because the self-assembly of flexible peptides is prone to diverge upon subtle structural change. To accomplish controllable morphology transformation, systematic self-assembly studies are performed using congener short β-peptide foldamers to find a minimal structural change that alters the self-assembled morphology. Introduction of oxygen-containing β-amino acid (ATFC) for subtle electronic perturbation on hydrophobic foldamer induces a previously inaccessible solid-state conformational split to generate the most susceptible modification site for morphology transformation of the foldamer assemblies. The site-dependent morphological switching power of ATFC is further demonstrated by dual substitution experiments and proven by crystallographic analyses. Stepwise morphology transformation is shown by modifying an identical foldamer scaffold. This study will guide in designing peptidic molecules from scratch to create complex and biofunctional assemblies with nonspherical shapes.

Molecular engineering and modification of FCC slurry oil residue for improving ageing resistance of high quality paving asphalt

In the present work, a novel resin molecule based on fluid catalytic cracking (FCC) slurry oil residue was designed and prepared with aid of molecular simulation to improve its ageing resistance. The average molecular structure of resin was characterized with elemental analysis, Fourier-transform IR (FT-IR) spectroscopy, NMR spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and designed resin was synthesized by attaching an additional propyl group to the as-received resin. The interactions of resins and asphaltene molecules were investigated via molecular simulation, UV-visible (UV-Vis) spectroscopy and atomic force microscopy (AFM). The results revealed that the equilibrium distance of designed resin-asphaltene dimer decreased from 0.42 nm to 0.38 nm while the lowest contact energy increased from 9.3 kcal/mol to 11.2 kcal/mol, indicating an improved stability of designed resin-asphaltene particles. The adsorption isotherm indicated that more modified resin molecules could be adsorbed by asphaltene than the original resin. Compared with original asphalt, less aggregation of asphaltene occurred in asphalt blended with modified slurry oil residue during ageing process, showing its greatly improved ageing resistance.

Exploring of tumor-associated carbonic anhydrase isoenzyme IX and XII inhibitory effects and cytotoxicities of the novel N-aryl-1-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-1H-pyrazole-3-carboxamides

A series of novel N-aryl-1-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-1H-pyrazole-3-carboxamides was synthesized and examined as inhibitors of cytosolic (human) hCA I and hCA II, and cancer-related transmembrane hCA IX and hCA XII isoenzymes. AC2 was the most selective inhibitor towards cancer-related hCA IX while AC8 and AC9 selectively inhibited hCA XII over off-target isoenzymes. Anticancer effects of the compounds were evaluated towards human oral squamous cell carcinoma (OSCC) cell lines, human mesenchymal normal oral cells, breast (MCF7), prostate (PC3), non-small cell lung carcinoma cells (A549), and non-tumoral fetal lung fibroblast cells (MRC5). Compounds moderately showed cytotoxicity towards cancer cell lines. Among others, AC6 showed cell-specific cytotoxic activity and induced apoptosis in a dose-dependent manner without a significant change in the cell cycle distribution of MCF7. These results suggest that pyrazole-3-carboxamides need further molecular modification to increase their anticancer drug candidate potency.

Functional dissection and modulation of the BirA protein for improved autotrophic growth of gas-fermenting Clostridiumljungdahlii.

SummaryGas‐fermenting Clostridium species can convert one‐carbon gases (CO2/CO) into a variety of chemicals and fuels, showing excellent application prospects in green biological manufacturing. The discovery of crucial genes and proteins with novel functions is important for understanding and further optimization of these autotrophic bacteria. Here, we report that the Clostridium ljungdahlii BirA protein (ClBirA) plays a pleiotropic regulator role, which, together with its biotin protein ligase (BPL) activity, enables an effective control of autotrophic growth of C. ljungdahlii. The structural modulation of ClBirA, combined with the in vivo and in vitro analyses, further reveals the action mechanism of ClBirA’s dual roles as well as their interaction in C. ljungdahlii. Importantly, an atypical, flexible architecture of the binding site was found to be employed by ClBirA in the regulation of a lot of essential pathway genes, thereby expanding BirA’s target genes to a broader range in clostridia. Based on these findings, molecular modification of ClBirA was performed, and an improved cellular performance of C. ljungdahlii was achieved in gas fermentation. This work reveals a previously unknown potent role of BirA in gas‐fermenting clostridia, providing new perspective for understanding and engineering these autotrophic bacteria.

Control strategies for the vertical gene transfer of quinolone ARGs in Escherichia coli through molecular modification and molecular dynamics

This study investigates the regulation of the vertical gene transfer of quinolones’ antibiotic resistance genes (ARGs) through a combination of source modification and process control. In source prevention, 29 Escherichia coli (E. coli) DNA gyrase subunit A mutant proteins were constructed, the B-G mutant protein displayed the greatest reduction in binding effect (−25.98%). Based on this, a 3D-QSAR model was constructed, and LEV-2 and LEV-9 QNs derivatives were designed based on Levofloxacin (LEV), and their binding effect with B-G mutant protein was found be increased by 13.24% and 19.40%. The drug resistance mechanism of E. coli was explored based on molecular docking technology and protein hydrophobic interaction theory. Most of the amino acid resistance mutations changed from hydrophilic to lipophilic, which inhibited the binding of QNs to mutant protein A subunit, and further reduced the bactericidal effect of QNs. In process control, Huoxiang-Zhengqi, stroke-physiological saline solution (SPSS), and Lycium barbarum (L. barbarum) was found to be 164.82% higher than that of the blank control group. The purpose of this study is to provide a theoretical support for the joint regulation of QNs’ ARGs in organisms and the research and development on green alternatives to QNs compounds.

The Influence of Various Growth Regulators on Induction Organogenic Callus from Gajah and Kuning Cassava Genotype (Manihot esculenta Crantz)

Kuning and Gajah genotypes are two collections of cassava in the Biotechnology Research Center for Germplasm, LIPI with the advantages of each genotype are high beta carotene and high production. The multiplication in in vitro culture can be done one of them through organogenesis. The aim of this study was to evaluate the effect of using 2,4-D; NAA and Kinetin are used singly for the formation of organogenesis of cassava in the Kuning Cassava and Gajah genotypes. This research was conducted at the Laboratory of Molecular Genetics and Modification of Plant Biosynthetic Pathways, Biteknologi Research Center, LIPI, Bogor since January - February 2018. The source of explants were young leaves and petiols from cassava plant culture in vitro genotypes of Gajah and Kuning yam which were three months old. in culture. The basic media used as a planting medium were Murashige and Skoog (MS) media with the addition of growth regulators (ZPT) singly, 2,4-D, NAA and Kinetin with two concentrations of ZPT each, 8 and 10 mg L- 1 This research was arranged based on a completely randomized design factorial pattern consisting of 2 factors. All data obtained were analyzed using ANOVA and if there is an influence then proceed with the DMRT test with an error rate of 5% using the SPSS program. The highest number of Kuning genotype cassava organogenic callus that developed into shoots on the medium added by ZPT was 2.4 D and kinetin with the same concentration of 8 mg L-1. Formation of the best organogenic callus in petiol explants in the media with the addition of a single 2,4-D and Kinetin with the same concentration of 8 mg L-1. Keywords: Cassava, growth regulators, organogenic.