Amidase Production Research Articles

Improvement of amidase production with high specific acyltransferase activity using Bacillus smithii IITR6B2

This work introduces an approach to increase the productivity of the whole-cell amidase's acyltransferase activity of the Bacillus smithii IITR6b2 in terms of its specific activity. Amidase has a wide range of substrate specificities. An increased amidase with acyl transfer activity will help produce an abundance of different hydroxamates and acid hydrazides with only a small amount of biocatalyst. Unlike the chemical process, it involves a greener approach using an aqueous media and one-step biotransformation. The statistical optimization of amidase production was done through Plackett-Burman design and Response Surface Methodology. After the screening, the ANOVA showed a p-value of <0.001 with a determination coefficient (R2) of 0.994. The four critical factors selected from the Plackett-Burman design were yeast extract, sodium chloride, manganese chloride, and trisodium citrate. After the Central Composite Design, the ANOVA revealed a p-value of <0.001 and a determination coefficient (R2) of 0.9887. The overall enzyme activity increased from 18 IU/ml to 91.30 IU/ml (a 5-fold increase), and specific enzyme activity from 16.1 IU/mg to 47.66 IU/mg (a nearly 3-fold increase). After validation of the predicted model from Response Surface Methodology, yeast extract had the maximum influence on amidase production and activity in combination with manganese chloride. Further, the produced enzyme was explored for the synthesis of pyrazine-2-carboxylic acid hydrazide in a solvent-free reaction system.

Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering

A highly active secretory expression system in Bacillus subtilis was constructed for enhancing the heterogeneous secretory expression efficiency of a recombinant amidase from Bacillus megaterium (Bm-Ami). Initially, six promoters PlytR, PspoVG, PaprE, PyvyD, PftsH, PamyE were screened out from 33 endogenous phase-dependent promoters via the strategy of promoter engineering which showed higher transcriptional levels than the original promoter Pcdd. Afterwards, a secretory expression system pBSHdd2-20 containing a dual-promoter PamyE-cdd and a signal peptide Pac was constructed showing an extracellular Bm-Ami activity of 135.58 U mL−1 during shake-flask cultivation, which was 3.58-fold greater than that with plasmid pBSH1 (control). Finally, extracellular amidase activity of a strain containing the plasmid pBSHdd2-20 reached 430 U mL−1 (equal to 10.8 mg mL−1) and 10.72 U mg−1DCW after 52 h in a scale-up fermentation, which was the highest secretory efficiency for Bm-Ami in all studies to date. In conclusion, a highly secretory system constructed in this study can be applied for the industrial production of amidase and provides a basis for the development of an excellent secretory expression system for similar proteins.

Multi-objective based superimposed optimization method for enhancement of l-glutaminase production by Bacillus subtilis RSP-GLU

l-Glutaminase (l-glutamine amidohydrolase, EC 3.5.1.2) is a deamiding enzyme that has current focus on a therapeutic potentials and flavor enhancement agent in food industry. It was noticed that the amidase production was depended on the bacterial growth. However, the enhancement of bacterial growth by other substrates suppress the secretion of l-glutaminase. Governing the growth as well as amidase production is challenging task. In this study a multi-objective based superimposed optimization method was employed to control the biomass as well as to enhance the production of l-glutaminase. Various parameters such as volume of medium, pH, incubation temperature, inoculum level, l-glutamine concentration and rpm were optimized. The interactive influence of these parameters with their effect on enzyme and biomass production was analyzed using fractional factorial central composite design (FFCCD). The coefficient of determination (R2) was calculated as 0.9862 and 0.9513 for l-glutaminase and biomass production respectively. Overlay graphs were plotted by superimposing the contours for the various response surfaces of biomass and enzyme production. Based on superimposing method the cultural conditions obtained was volume of media 85 mL, temperature 37 °C, pH 7.24, inoculum concentration 2.47 mL, l-glutaminase concentration 1.01 g/L and agitation speed of 109 rpm. At these conditions 225 U/mL l-glutaminase and 0.79 g/L biomass were obtained. The current study provides a good description for balanced optimization of fermentation process of multi response or objective systems by using FFCCD method.

Enhanced production of thermostable amidase from Geobacillus subterraneus RL-2a MTCC 11502 via optimization of physicochemical parameters using Taguchi DOE methodology.

The specific effect of chemical and physical factors on amidase production from Geobacillus subterraneus RL-2a was investigated using design of experiments (DOE) methodology. The one-factor-at-a-time (OFAT) method was used to study the effects of carbon and nitrogen sources on amidase production. Subsequently, optimal levels of physical parameters and key media components, namely temperature, pH, sucrose, K2HPO4, NaCl, yeast, CaCl2·2H2O and MgSO4·7H2O, were determined using the Taguchi orthogonal array (OA) experimental design (DOE) methodology. Taguchi method based on three levels with a OA layout of L18 (21 × 37) with eight most influential factors on amidase synthesis for the proposed experimental design. Analysis of variance was performed on the obtained results and optimum condition suggested by statistical calculations was tested in a verification test. An increase of 169.56 % in amidase production compared to the unoptimized conditions was observed and the conversion of isonicotinamide was significantly improved after performing optimization techniques, including OFAT and Taguchi method. The result indicated that Taguchi method was effective in optimizing the culture conditions of amidase production.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0390-1) contains supplementary material, which is available to authorized users.

Production optimization of extracellular amidase enzyme by newly isolated Pseudomonas putida AP-2 from agricultural soil

Amidases are ubiquitous enzymes that have received increased attention due to their wide range of biotechnological applications, especially in industries for the synthesis of wide variety of carboxylic and hydroxamic acids, which find applications in pharmaceuticals, agrochemicals and waste water treatments. In the present study, 40 bacterial isolates were screened for extracellular amidase-producing capability, and on the basis of color development, 5 isolates were selected for amidase production in broth media. Based on enzyme production, one of the most potent isolates identified as Pseudomonas putida AP-2 was selected for further study. The effects of media composition and various fermentation conditions for optimization of amidase production were studied. The maximum extracellular amidase production was obtained at 30 °C and pH 8.0 after 36 h of incubation in shaking condition. Among the substrate, acetamide was the best; however, P. putida AP-2 also utilized acrylamide which is a known carcinogen. Regarding carbon sources, glucose was the best, while peptone was found the best nitrogen source. The isolated bacterium, P. putida AP-2, is also tolerant to number of heavy metals at higher levels so this may also be applied for field application in contaminated soil.

Production of l-tryptophan by enantioselective hydrolysis of d,l-tryptophanamide using a newly isolated bacterium

AbstractBacterial strain ZJB-09211 capable of amidase production has recently been isolated from soil samples. The strain is able to asymmetrically hydrolyze l-tryptophanamide from d,l-tryptophanamide to produce l-tryptophan in high yield and with excellent stereoselectivity (enantiomeric excess &gt; 99.9 %, and enantiomeric ratio &gt; 200). Strain ZJB-09211 has been identified as Flavobacterium aquatile based on the cell morphology analysis, physiological tests, and the 16S rDNA sequence analysis. Optimization of the fermentation medium led to an about six-fold increase in the amidase activity of strain ZJB-09211, which reached 501.5 U L−1. Substrate specifity and stereoselectivity investigations revealed that amidase of F. aquatile possessed a broad substrate spectrum and high enantioselectivity.

A comparative study on the production of amidase using immobilized and dehydrated immobilized cells of Pseudomonas putida MTCC 6809

Pseudomonas putida MTCC 6809, a plant growth promoting rhizobacteria producing amidase was isolated from the rhizosphere of Pisum sativum. The cells were immobilized in sodium alginate for the production of amidase and the effect of dehydration on immobilized beads were studied. Optimization of process parameters for amidase production was carried out to enhance enzyme production using immobilized cells. From the results it is clear that 2% and 3% (w/v) of alginate were suitable for amidase production with 12.8 and 13 U/ml activity, respectively after 36 h of incubation. Among the various substrates studied acetamide (2% w/v) was a good inducer of amidase. It was observed that immobilized catalysts could be recycled up to five batches. Amidase production was observed in both free and immobilized cells, nevertheless immobilization is much favored in comparison to free cells, as it leads to reusability of beads, lesser contamination, consistent amidase production and adaptability to wide range of culture conditions. The relative enzyme activity with the dehydrated beads was only 27% in comparison to hydrated beads, it is possible to pack considerably more into a fixed volume as the relative volume of dehydrated beads is 20%. Even though consistent amidase production was difficult to achieve using dehydrated beads, which may have certain advantages like less chances for microbial contamination and easy to transport.

Hyper production of enzyme penicillin amidase by locally isolated thermo-tolerantBacillus sp. MARC-0103 from rice starch in cheese whey

The present study concern with the extracellular production of penicillin amidase in a cost-effective cheese whey medium under submerged fermentation. ABacillus sp. MARC-0103 producing a high level of extra cellular penicillin G amidase was isolated from rice starch by heat shock method. The penicillin G amidase production in the strain was induced by phenyl acetic acid. The culture medium was optimized by using Plackett-Burman and central composite experimental designs for enhanced production of penicillin amidase. The factorial design indicated that the main factors that positively affect penicillin amidase production were casein hydro-lysate, CaCl2·2H2O, FeCI3·6H2O, Na2SO4 and cheese whey, whereas the presence of calcium carbonate and magnesium chloride in the medium had no effect on enzyme production. Phenyl acetic acid concentration and time of addition was found critical for enzyme pro duction. Enzyme production was enhanced very much by multiple addition of inducer. Other cultural condition such as pH, temperature, inoculum size and age were also optimized. More than two fold increase in enzyme production (40.7 U/ml/min) was observed under optimized cultural conditions. The molecular mass was estimated to be 40.0 kDa by SDS-PAGE.

Enhanced production of amidase from Rhodococcus erythropolis MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket-Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.

Ca2+ Is a Cofactor Required for Membrane Transport and Maturation and Is a Yield-Determining Factor in High Cell Density Penicillin Amidase Production

Penicillin amidases (PAs) from E. coli and A. faecalis are periplasmic enzymes that contain one tightly bound Ca(2+) per molecule that does not directly participate in the enzymatic function. This ion may, however, be required for the maturation of the pre-pro-enzyme. The pro-enzyme of homologous PAs are translocated through the Tat- (E. coli PA(EC)) and Sec- (A. faecalis PA(AF)) transport systems, respectively. Cell fractionation, electrophoresis, immunoblotting, and activity staining demonstrated that Ca(2+) binding is required for the membrane transport and maturation of the pro-enzyme to active enzyme. Pro-enzyme without Ca(2+) was targeted to the membrane but not translocated. Influence of Ca(2+) in medium and feed was studied for high cell density cultivations of E. coli expressing these enzymes. Without Ca(2+) in the feed the synthesis of the pre-pro-enzyme was hardly influenced. At optimal Ca(2+) content in the feed the active enzyme amount could be increased by 2 orders of magnitude up to 0.9 g/L (PA(EC)) and 2.3 g/L (PA(AF)) or 4% (PA(EC)) and 8% (PA(AF)) of the cell dry weight. The corresponding specific activities are 1700 U (PA(EC)) and 14000 U (PA(AF)) per gram cell dry weight, respectively. These values are higher than those published previously. Thus, for optimal yields of the studied and other extra- and periplasmic enzymes that require Ca(2+) or other ions as cofactors for membrane transport and maturation, sufficient cofactor must be added in the feed.

Physiology of amidase production by M. methylotrophus: Isolation of hyperactive strains using continuous culture

Physiology of amidase production by M. methylotrophus: Isolation of hyperactive strains using continuous culture

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium

BackgroundDuring the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved.ResultsFor this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached.ConclusionThe combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations.

Comparative Study of Amidase Production by Free and Immobilized &lt;I&gt;Escherichia coli&lt;/I&gt; Cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30 degrees C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.

Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa

Vfr of Pseudomonas aeruginosa is 91% similar to the cAMP receptor protein (CRP) of Escherichia coli. Based on the high degree of sequence homology between the two proteins, the question arose whether Vfr had a global regulatory effect on gene expression for P. aeruginosa as CRP did for E. coli. This report provides two-dimensional polyacrylamide gel electrophoretic evidence that Vfr is a global regulator of gene expression in P. aeruginosa. In a vfr101::aacC1 null mutant, at least 43 protein spots were absent or decreased when compared to the proteome pattern of the parent strain. In contrast, 17 protein spots were absent or decreased in the parent strain when compared to the vfr101::aacC1 mutant. Thus, a mutation in vfr affected production of at least 60 proteins in P. aeruginosa. In addition, the question whether Vfr and CRP shared similar mechanistic characteristics was addressed. To ascertain whether Vfr, like CRP, can bind cAMP, Vfr and CRP were purified to homogeneity and their apparent dissociation constants (K(d)) for binding to cAMP were determined. The K(d) values were 1.6 microM for Vfr and 0.4 microM for CRP, suggesting that these proteins have a similar affinity for cAMP. Previously the authors had demonstrated that Vfr could complement a crp mutation and modulate catabolite repression in E. coli. This study presents evidence that Vfr binds to the E. coli lac promoter and that this binding requires the presence of cAMP. Finally, the possible involvement of Vfr in catabolite repression control in P. aeruginosa was investigated. It was found that succinate repressed production of mannitol dehydrogenase, glucose-6-phosphate dehydrogenase, amidase and urocanase both in the parent and in two vfr null mutants. This implied that catabolite repression control was not affected by the vfr null mutation. In support of this, the cloned vfr gene failed to complement a mutation in the P. aeruginosa crc gene. Thus, although Vfr is structurally similar to CRP, and is a global regulator of gene expression in P. aeruginosa, Vfr is not required for catabolite repression control in this bacterium.

Transcriptional analysis of the nitrile-degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli-T7 expression system.

Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70-like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli-T7 expression system by lowering the growth temperature to 29 degrees C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximately 1:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.

Polyacrylamide as a substrate for microbial amidase in culture and soil

High molecular weight, linear polyacrylamide (PAM) with anionic charge is added to agricultural soils as an anti-erosion additive. Research indicates that soil microorganisms are able to utilize PAM as a source of N and that inorganic N pools are altered in some PAM-treated soils. The potential role of hydrolytic amidase activity in the microbial utilization of PAM for N was investigated. Intracellular and extracellular amidase activity was measured over time in enrichment cultures which used PAM as sole N source. Enzyme activity increased concomitant with cell growth and N removal from PAM. Cell growth, N removal and amidase production were dependent upon readily-available C in the medium. Amidase activity and substrate specificity were determined for PAM-utilizing enrichment cultures exposed to various N sources. Polyacrylamide-specific amidase activity appears to be inducible, and not constitutive, based on the lack of amidase activity in cultures supplied with only ammonium nitrate for N versus substantial activity when PAM was added as an amendment with or without ammonium nitrate. Cultures amended with propionamide exhibited amidase activity largely specific for this small amide substrate, while cultures supplied with PAM as sole N source exhibited amidase activity specific for formamide, propionamide and PAM. Amidase activity and substrate specificity were determined for PAM-treated and untreated agricultural field soils. Polyacrylamide-specific amidase activity was higher in PAM-treated soil (14.86±14.0 μg NH 4 + released g −1 soil) than in untreated soil (1.02±2.3 μg NH 4 + released g −1 soil); activity specific for low molecular weight amides was slightly elevated or unchanged in PAM-treated soil as compared with untreated soil.

Optimization of culture conditions of Brevibacterium sp. for the production of amidase and adipamidase

The culture conditions required for the maximum production of the wide spectrum amidase and the adipamidase were determined for two strains of Brevibacterium sp. applying two mathematical techniques: the Hadamard matrix and the analyses of response surface. A culture medium was optimized and the role of Zn 2+ on the activity of adipamidase was demonstrated. The enzyme was shown to be not subject to catabolic repression by glucose or ammonium sulfat and it was not inducible. The influence of physical and chemical factors (pH, temperature) on cell growth and enzyme activity was also studied.

Improved penicillin amidase production using a genetically engineered mutant of Escherichia coli ATCC 11105

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.

Effect of recycling of fermentation broth for the production of penicillin amidase

In general in any fermentation the volume of wastewater is too high. The excess water might be utilized to reduce consumption of process water. In this investigation cell-free culture filtrate was used instead of process water. In batch experiments, cell-free culture filtrate was used in ratios between 0·1 and 1·0 for eight subsequent batches. The specific growth rate was higher than that of the first batch for reusability ratios between 0·1 and 0·8 in all batches. Synthesis of penicillin amidase was found to be more suitable in the fourth batch with a recycle ratio between 0·6 and 1·0. An extracellular protein factor was found to influence the synthesis of penicillin amidase.

Physiology of Amidase Production by Methylophilus methylotrophus: Isolation of Hyperactive Strains Using Continuous Culture

The obligately methylotrophic bacterium Methylophilus methylotrophus hydrolyses aliphatic amides to ammonia and aliphatic acid using a cytoplasmic amidase. Physiological regulation of amidase activity was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. The results showed that synthesis of the enzyme was induced by various amides (acrylamide &gt; acetamide) and repressed by ammonia. Growth of the wild-type organism in acetamide-limited continuous culture at very low dilution rate (D 0·025 h−1) led to the selection of a hyperactive strain (MM6), the subsequent growth of which under acrylamide limitation led to the selection of another strain (MM8) which showed even higher activity. The amidase activities of strains MM6 and MM8 were respectively approximately four and twelve times higher than that of the wild-type organism following growth under similar conditions, whereas the concentrations of the enzyme as determined by SDS-PAGE and scanning densitometry were approximately four times higher than the wild-type organism in both strains. The amidase in strain MM8 exhibited a K m for acrylamide that was approximately one-third lower than that of the wild-type organism or of strain MM6. It is concluded that the hyperactivity of strain MM6 was due predominantly to the production of more wild-type enzyme, whilst the hyperactivity of strain MM8 was due to the production of approximately the same amount of enzyme as strain MM6 (up to 25% of the total cell protein depending on the nature of the limiting amide) but with a substantially enhanced catalytic activity (K cat). These changes were apparently the result of spontaneous mutations that occurred in response to growth at extremely low amide concentrations, giving the novel strains a strong selective advantage under these conditions (possibly by enhancing the rate of diffusion of amide into the cell).