Improvement of the activity of l-asparaginase I improvement of the catalytic activity of l-asparaginase I from Bacillus megaterium H-1 by in vitro directed evolution

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate.

Download Full-text

OP0039 ALPN-303, AN ENHANCED, POTENT DUAL BAFF/APRIL ANTAGONIST ENGINEERED BY DIRECTED EVOLUTION FOR THE TREATMENT OF SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND OTHER B CELL-RELATED AUTOIMMUNE DISEASES

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.727 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 21.2-21

Author(s):

S. R. Dillon ◽

L. S. Evans ◽

K. E. Lewis ◽

J. Yang ◽

M. W. Rixon ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Autoimmune Diseases ◽

B Cell ◽

Directed Evolution ◽

Human Lymphocyte ◽

Lupus Erythematosus ◽

Plasma Cells ◽

Systemic Lupus ◽

Cynomolgus Monkeys

Background:BAFF and APRIL are TNF superfamily members that form homo- and heteromultimers that bind TACI and BCMA on B cells; BAFF also binds BAFF-R. BAFF and APRIL support B cell development, differentiation, and survival, particularly for plasmablasts and plasma cells, and play critical roles in the pathogenesis of B cell-related autoimmune diseases. In nonclinical models, inhibition of either BAFF or APRIL alone mediates relatively modest effects, whereas their co-neutralization dramatically reduces B cell function, including antibody production. Fc fusions of wild-type (WT) TACI (e.g. atacicept and telitacicept) target both BAFF and APRIL and have demonstrated promising clinical potential in e.g. systemic lupus erythematosus (SLE) and IgA nephropathy but have not yet clearly exhibited long-term and/or complete disease remissions.Objectives:To generate a dual BAFF/APRIL antagonist with inhibitory activity superior to WT TACI and BCMA and with the potential to improve clinical outcomes in B cell-mediated diseases.Methods:Our directed evolution platform was used to identify a potent variant TNFR domain (vTD) of TACI that exhibits significantly enhanced affinity for BAFF and APRIL as compared to WT TACI; this TACI vTD domain was fused to a human IgG Fc to generate the therapeutic candidate ALPN-303. ALPN-303 was evaluated for functional activity in: 1) human lymphocyte assays, 2) the NOD.Aec1Aec2 spontaneous model of Sjogren’s syndrome (SjS), 3) the bm12-induced mouse model of lupus, 4) the (NZB/NZW)F1 spontaneous model of lupus, and 5) preclinical rodent and cynomolgus monkey pharmacokinetic/pharmacodynamic studies.Results:ALPN-303 inhibited BAFF- and APRIL-mediated signaling in vitro in human lymphocyte assays, with significantly lower IC50 values than WT TACI-Fc and belimumab comparators. In all mouse models evaluated, administration of ALPN-303 rapidly and significantly reduced key lymphocyte subsets including plasma cells, germinal center B cells, and follicular T helper cells. ALPN-303 significantly reduced autoantibodies and sialadenitis in the spontaneous SjS model, inhibited glomerular IgG deposition in the bm12-induced model of lupus, and potently suppressed anti-dsDNA autoAbs, blood urea nitrogen levels, proteinuria, sialadenitis, kidney lesions, and renal immune complex deposition in the NZB/W lupus model. As compared to WT TACI-Fc, ALPN-303 exhibited higher serum exposure and significantly and persistently decreased titers of serum IgM, IgG, and IgA antibodies in mice and cynomolgus monkeys (Figure 1).Figure 1.ALPN-303 induces more potent suppression, as compared to WT TACI-Fc, of serum immunoglobulins following a single 9 mg/kg IV infusion (on Day 0; arrows) in female cynomolgus monkeys.Conclusion:ALPN-303 is a potent BAFF/APRIL antagonist derived from our directed evolution platform that consistently demonstrates encouraging immunomodulatory activity and efficacy in vitro and in vivo, superior in preclinical studies to anti-BAFF antibody and WT TACI-Fc. This novel Fc fusion molecule demonstrates favorable preliminary developability characteristics, including higher serum exposures and more potent immunosuppressive activities, which may enable lower clinical doses and/or longer dosing intervals than WT TACI-Fc therapeutics. ALPN-303 may thus be an attractive development candidate for the treatment of multiple autoimmune and inflammatory diseases, particularly B cell-related diseases such as SLE, SjS, and other connective tissue diseases. Preclinical development is underway to enable the initiation of clinical trials later this year.Disclosure of Interests:Stacey R. Dillon Shareholder of: Alpine Immune Sciences, Bristol Myers Squibb, Employee of: Alpine Immune Sciences, Bristol Myers Squibb, Lawrence S. Evans Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Katherine E. Lewis Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Jing Yang Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Mark W. Rixon Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Joe Kuijper Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Dan Demonte Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Janhavi Bhandari Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Steve Levin Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Kayla Kleist Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Sherri Mudri Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Susan Bort Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Daniel Ardourel Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Michelle A. Seaberg Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Rachel Wang Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Chelsea Gudgeon Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Russell Sanderson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Martin F. Wolfson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Jan Hillson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Stanford L. Peng Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences

Download Full-text

Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

Download Full-text

Random-priming in vitro recombination: An effective tool for directed evolution

Nucleic Acids Research ◽

10.1093/nar/26.2.681 ◽

1998 ◽

Vol 26 (2) ◽

pp. 681-683 ◽

Cited By ~ 104

Author(s):

Z. Shao ◽

H. Zhao ◽

L. Giver ◽

F. H. Arnold

Keyword(s):

Directed Evolution ◽

In Vitro Recombination ◽

Random Priming

Download Full-text

The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

The Journal of Cell Biology ◽

10.1083/jcb.201011126 ◽

2011 ◽

Vol 193 (6) ◽

pp. 1009-1020 ◽

Cited By ~ 33

Author(s):

Martijn Gloerich ◽

Marjolein J. Vliem ◽

Esther Prummel ◽

Lars A.T. Meijer ◽

Marije G.A. Rensen ◽

...

Keyword(s):

Catalytic Activity ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Negative Regulator ◽

Camp Signaling ◽

Nuclear Pore ◽

Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Wide Range

Cyclic adenosine monophosphate (cAMP) is a second messenger that relays a wide range of hormone responses. In this paper, we demonstrate that the nuclear pore component RanBP2 acts as a negative regulator of cAMP signaling through Epac1, a cAMP-regulated guanine nucleotide exchange factor for Rap. We show that Epac1 directly interacts with the zinc fingers (ZNFs) of RanBP2, tethering Epac1 to the nuclear pore complex (NPC). RanBP2 inhibits the catalytic activity of Epac1 in vitro by binding to its catalytic CDC25 homology domain. Accordingly, cellular depletion of RanBP2 releases Epac1 from the NPC and enhances cAMP-induced Rap activation and cell adhesion. Epac1 also is released upon phosphorylation of the ZNFs of RanBP2, demonstrating that the interaction can be regulated by posttranslational modification. These results reveal a novel mechanism of Epac1 regulation and elucidate an unexpected link between the NPC and cAMP signaling.

Download Full-text

pH-induced hysteretic properties of phosphofructokinase purified from rat myocardium

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1986.250.3.r505 ◽

1986 ◽

Vol 250 (3) ◽

pp. R505-R511 ◽

Cited By ~ 2

Author(s):

S. C. Hand ◽

J. F. Carpenter

Keyword(s):

Catalytic Activity ◽

Metabolic Regulation ◽

Inverse Correlation ◽

Rat Myocardium ◽

Solution Ph ◽

Reversible Inactivation ◽

Ph Range ◽

Hysteretic Loss ◽

Fructose 1,6 Bisphosphate

Phosphofructokinase (PFK) purified from the rat myocardium is reversibly inactivated under a pH regime approximating that reported for ischemic hearts. At pH 6.5 and 37 degrees C, the enzyme displays a hysteretic loss of activity during 60-min incubations, declining to 48% of control (pH 7.1, 37 degrees C) values. Citric acid increases the degree of inactivation (28% of control), whereas fructose 1,6-bisphosphate reduces the decline in activity. Simultaneous measurements of 90 decreases light scattering and catalytic activity suggest the inactivation is temporally linked to dissociation of active tetrameric enzyme into an inactive form of lower molecular weight. Fluorescence enhancement of the extrinsic probe sodium mansate, which binds preferentially to dimeric PFK, indicates that the equilibrium dimer concentration (cp1 infinity) increases as pH is lowered. This increase in cp1 infinity exhibits a strong inverse correlation (r = 0.984) with catalytic activity across the pH range of 8.0 to 6.5. Returning solution pH to 7.0 or above promotes a time-dependent reactivation and repolymerization of PFK. The rate of reactivation is increased at higher enzyme concentrations and in the presence of trimethylamine-N-oxide, a nitrogenous osmolyte noted for its ability to promote protein aggregation reactions. Thus these results demonstrate the capacity of rat heart PFK to undergo reversible inactivation and dissociation in vitro and represent the first phase of a two-part study testing the hypothesis that these pH-induced hysteretic processes are operative in the ischemic myocardium. The data are evaluated in terms of the potential roles of hysteretic enzymes in metabolic regulation.

Download Full-text

Small–molecule therapeutics for the treatment of glycolipid lysosomal storage disorders

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1278 ◽

2003 ◽

Vol 358 (1433) ◽

pp. 927-945 ◽

Cited By ~ 52

Author(s):

Terry D. Butters ◽

Howard R. Mellor ◽

Keishi Narita ◽

Raymond A. Dwek ◽

Frances M. Platt

Keyword(s):

Catalytic Activity ◽

Lysosomal Storage Disorders ◽

Substrate Reduction Therapy ◽

Lysosomal Storage ◽

Substrate Reduction ◽

Treatment Demand ◽

Small Molecule Therapeutics ◽

Storage Disorders

Glycosphingolipid (GSL) lysosomal storage disorders are a small but challenging group of human diseases to treat. Although these disorders appear to be monogenic in origin, where the catalytic activity of enzymes in GSL catabolism is impaired, the clinical presentation and severity of disease are heterogeneous. Present attitudes to treatment demand individual therapeutics designed to match the specific disease–related gene defect; this is an acceptable approach for those diseases with high frequency, but it lacks viability for extremely rare conditions. An alternative therapeutic approach termed ‘substrate deprivation’ or ‘substrate reduction therapy’ (SRT) aims to balance cellular GSL biosynthesis with the impairment in catalytic activity seen in lysosomal storage disorders. The development of N–alkylated iminosugars that have inhibitory activity against the first enzyme in the pathway for glucosylating sphingolipid in eukaryotic cells, ceramide–specific glucosyltransferase, offers a generic therapeutic for the treatment of all glucosphingolipidoses. The successful use of N–alkylated iminosugars to establish SRT as an alternative therapeutic strategy has been demonstrated in in vitro , in vivo and in clinical trials for type 1 Gaucher disease. The implications of these studies and the prospects of improvement to the design of iminosugar compounds for treating Gaucher and other GSL lysosomal storage disorders will be discussed.

Download Full-text

Promoción de crecimiento en trigo (Triticum turgidum L. subsp. durum) por la co-inoculación de cepas nativas de Bacillus aisladas del Valle del Yaqui, México

Nova Scientia ◽

10.21640/ns.v12i24.2136 ◽

2020 ◽

Vol 12 (24) ◽

Author(s):

Jonathan Rojas Padilla ◽

Luis Abraham Chaparro Encinas ◽

Rosa Icela Robles Montoya ◽

Sergio De los Santos Villalobos

Keyword(s):

Bacillus Megaterium ◽

Triticum Turgidum ◽

Bacillus Paralicheniformis

Introducción: Las rizobacterias promotoras del crecimiento vegetal (RPCV) son un grupo de bacterias rizosféricas con la habilidad de promover el crecimiento y la salud de las plantas, y restaurar la fertilidad del suelo. El presente estudio tuvo como objetivo evaluar la promoción del crecimiento en el cultivo de trigo (Triticum turgidum L. subsp. durum) por la co-inoculación de cepas nativas del género Bacillus aisladas del Valle del Yaqui, México, para su potencial uso como inoculante microbiano.Método: Tres cepas bacterias obtenidas de la Colección de Microorganismos Edáficos y Endófitos Nativos (COLMENA), aisladas del cultivo de trigo en el Valle del Yaqui, fueron estudiadas. Primeramente, se realizó la identificación molecular de las cepas mediante la secuenciación del gen 16S RNAr, mediante la plataforma Sanger. Además, las cepas bacterianas fueron caracterizadas metabólicamente mediante actividades funcionales asociadas a la promoción del crecimiento vegetal (producción de indoles, solubilización de fósforo insoluble, y producción de sideróforos). Finalmente, el impacto de la inoculación de estas cepas individualmente, y en consorcios fue determinado utilizando el cultivo de trigo (Triticum turgidum L subsp. durum) como planta modelo, simulando las condiciones edafo-climáticas del Valle del Yaqui. Las variables morfométricas medidas fueron la longitud aérea y de raíz, peso seco aéreo y de raíz, e índice de biovolumen.Resultados: Las cepas TRQ8, TRQ65 y TE3T fueron afiliadas taxonómicamente a Bacillus megaterium, Bacillus paralicheniformis y Bacillus cabrialesii, respectivamente; dicha clasificación fue soportada por sus características macro-microscópica como: su forma bacilar y tinción Gram positiva, que son características propias de este género bacteriano. Las cepas en estudio tuvieron la capacidad de producir indoles, siendo B. paralicheniformis TRQ65 quien presentó mayor producción con 39.29 µg/mL. Mientras que en la prueba de solubilización de fósforo insoluble las 3 cepas mostraron la capacidad en un rango de índice de solubilización de 1.37 a 1.43; finalmente, solamente la cepa B. megaterium TRQ8 mostró un índice de producción de sideróforos de 8.17. La inoculación del consorcio B. megaterium TRQ8 + B. paralicheniformis TRQ65 mostró los mayores incrementos en las 5 variables medidas en la planta, diferencia significativa (p<0.05) vs. el tratamiento no inoculado, la longitud aérea y radical mostró un incremento de 6 y 10% respectivamente, mientras que la biomasa seca aérea aumentó 60% y la biomasa seca radicular se incrementó en 82%. El índice de biovolumen se incrementó en 18% por la inoculación de dicho consorcio bacteriano.Discusión o Conclusión: Las cepas estudiadas presentan características de promoción de crecimiento in vitro e in vivo. Sin embargo, la co-inoculación de B. megaterium TRQ8 y B. paralicheniformis TRQ65 incrementó su capacidad de promoción del crecimiento en plantas de trigo. Por lo cual, los mecanismos asociados a dicho efecto, así como sus funciones ecológicas e interacción con los factores bióticos y abióticos de los agro-sistemas deben ser estudiadas para su validación en diferentes agroecosistemas, antes de su utilización extensiva como un inoculante microbiano.

Download Full-text

Efektifitas Pestisida Biologis Bacillus Cereus dan Bacillus Megaterium. sebagai Pengendali Spodoptera litura Fabr (Lepidoptera: Noctuidae)

Jurnal Penelitian Pertanian Terapan ◽

10.25181/jppt.v17i2.294 ◽

2017 ◽

Vol 17 (2) ◽

Author(s):

I Made Indra Agastya ◽

Aminudin Afandhi ◽

Luqman Qurata Aini

Keyword(s):

Bacillus Cereus ◽

Bacillus Megaterium ◽

Spodoptera Litura ◽

Laboratory Studies ◽

Pests And Diseases ◽

Control Effectiveness ◽

Randomized Design ◽

Completely Randomized Design ◽

Lepidoptera Noctuidae

This research was conducted in vitro in the laboratory. Studies conducted in the Laboratory of Bacteriology Department of Plant Pests and Diseases of the Faculty of Agriculture, University of Brawijaya, on the effectiveness of the bacteria Bacillus cereus and Bacillus megaterium as biological pesticides controlling Spodoptera litura. The objective of this study was to determine the effectiveness of the bacteria Bacillus sp as biological control. Effectiveness pesticide was measured by testing the incubation period and mortality in larvae of S. litura instar 3. This study used a completely randomized design (CRD), conducted observations every 6 hours until the larvae dead. The results showed that the percentage of mortality of S. litura reached 94.66% compared to the control, while the incubation time of the bacteria B. cereus cause disease until 29.84 hours. B. cereus and B. megaterium have the ability to incubate third instar larvae of S. litura up to 29.84 hours and caused the death of larvae up to 94.66%. Keywords: Biological pesticides, Bacillus cereus, Bacillus megaterium, entomopatogen, Spodoptera litura

Download Full-text

In Vitro Hepatic Assessment of Cineole and Its Derivatives in Common Brushtail Possums (Trichosurus vulpecula) and Rodents

Biology ◽

10.3390/biology10121326 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1326

Author(s):

Ravneel R. Chand ◽

Mhairi Nimick ◽

Belinda Cridge ◽

Rhonda J. Rosengren

Keyword(s):

Catalytic Activity ◽

Management Strategies ◽

Brushtail Possum ◽

Trichosurus Vulpecula ◽

Cytochrome P450 3A ◽

Phascolarctos Cinereus ◽

Catalytic Activities ◽

Rats And Mice ◽

Drug Metabolising Enzymes

Folivore marsupials, such as brushtail possum (Trichosurus Vulpecula) and koala (Phascolarctos cinereus), can metabolise higher levels of dietary terpenes, such as cineole, that are toxic to eutherian mammals. While the highly efficient drug metabolising enzymes, cytochrome P450 3A (CYP3A) and phase II conjugating enzymes (UDP-glucuronosyltransferase, UGT), are involved in the metabolism of high levels of dietary terpenes, evidence for inhibitory actions on these enzymes by these terpenes is scant. Thus, this study investigated the effect of cineole and its derivatives on catalytic activities of hepatic CYP3A and UGT in mice, rats, and possums. Results showed that cineole (up to 50 µM) and its derivatives (up to 25 µM) did not significantly inhibit CYP3A and UGT activities in mice, rats, and possums (both in silico and in vitro). Interestingly, basal hepatic CYP3A catalytic activity in the possums was ~20% lower than that in rats and mice. In contrast, possums had ~2-fold higher UGT catalytic activity when compared to mice and rats. Thus, these basal enzymatic differences may be further exploited in future pest management strategies.

Download Full-text