Presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant Escherichia coli strain

A recombinant plasmid (pMK57) was constructed by cloning the Bacillus stearothermophilus alpha-amylase gene into pUC8; plasmid pMK79 was then derived from pMK57 by inserting the bacterial (Vitreoscilla) hemoglobin gene into the latter plasmid. Both pMK57 and pMK79 were transformed into Escherichia coli strain JM 103 to make strains MK57 and MK79, respectively. Both MK57 and MK79 produced alpha-amylase and MK79 produced hemoglobin. MK79 outgrew MK57 in shake flasks in LB medium, the advantage of the former appearing in late log phase. MK79 produced more alpha-amylase than MK57, on both per cell and per volume bases, in both mid and late log phases; the maximum advantage of MK79 (on a per volume basis) occurred in late log phase, at which time it produced 3.3 times as much alpha-amylase as MK57. The numbers of copies per cell of both pMK57 and pMK79 were significantly lower than that of pUC8.     

Selection and characterization of alpha-amylase-overproducing recombinant Escherichia coli containing the bacterial hemoglobin gene

We previously reported that the presence of the bacterial (Vitreoscilla) hemoglobin gene enhances alpha-amylase production in recombinant Escherichia coli strain MK79. Using the growth of MK79 on starch as a selective method we have produced a mutant strain (BSC9) that produces up to four times as much alpha-amylase as MK79. Both MK79 and BSC9 produce the most alpha-amylase (per cell and per milliliter) in the stationary phase; almost all of the enzyme is intracellular in both strains. Modification of the standard alpha-amylase assay increases the amount of amylase detected about sixfold. BSC9 has about five to nine times as many copies per cell as MK79 of the recombinant plasmid, which carries both the amylase and hemoglobin genes, but both strains produce about the same amount of hemoglobin. While MK79 respiration decreases upon going from log to stationary phase, BSC9 respiration increases during the same period. The two latter results may be of particular importance in determining the way in which hemoglobin enhances the production of cloned protein products in recombinant bacteria.Correspondence to: B. C. Stark     

A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10⁸ to 10⁹ per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the β-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.     

Specific Determination of alpha-Amylase Activity in Crude Plant Extracts Containing beta-Amylase

The specific measurement of α-amylase activity in crude plant extracts is difficult because of the presence of β-amylases which directly interfere with most assay methods. Methods compared in this study include heat treatment at 70°C for 20 min, HgCl₂ treatment, and the use of the α-amylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glycine max [L.] Merr.), and malted barley (Hordeum vulgare L.), the starch azure assay was the only satisfactory method for all tissues. While β-amylase can liberate no color alone, over 10 International units per milliliter β-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (about 4-fold) at β-amylase activities over 1,000 International units per milliliter. Two starch azure procedures were developed to eliminate β-amylase interference: (a) the dilution procedure, the serial dilution of samples until β-amylase levels are below levels that interfere; (b) the β-amylase saturation procedure, addition of exogenous β-amylase to increase endogenous β-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 International units per milliliter. These two procedures produced statistically identical results with most tissues, but not for all tissues. Differences between the two methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in β-amylase activity or inhibitory effects of the commercial β-amylase. The β-amylase saturation procedure was found to be preferable with most species. The heat treatment was satisfactory only for malted barley, as α-amylases in alfalfa and soybeans are heat labile. Whereas HgCl₂ proved to be a potent inhibitor of β-amylase activity at concentrations of 10 to 100 micromolar, these concentrations also partially inhibited α-amylase in barley malt. The reported α-amylase activities in crude enzyme extracts from a number of plant species are apparently the first specific measurements reported for any plant tissues other than germinating cereals.     

Thermostable α-amylase from derepressed Bacillus licheniformis produced in high yields from glucose

High yields of thermostable α-amylase was produced by Bacillus licheniformis 44MB82-G, resistant to glucose catabolite repression, on the basis of inexpensive raw materials and glucose as a main carbon source. The optimal parameters for the α-amylase production were an agitation rate of 500 rpm, constant air-flow rate (1 vvm) and cultivation temperature 40°C. An enzyme activity of 4800–5000 U/ml culture medium was reached in 96–120 h. The α-amylase preparation had the following characteristics: α-amylase activity 55 000 U/ml, high thermostability (98% residual α-amylase activity after 10 min treatment at 90°C), protein content 88 mg/ml and dry substances 30%.     

Antioxidant Status, α-Amylase Production,Growth, and Survival of Hemoglobin Bearing <Emphasis Type="Italic">Escherichia coli</Emphasis> Exposed to Hypochlorous Acid

In the present work, two matched strains of E. coli that bear a recombinant R-amylase gene (MK57) or the R-amylase gene and vgb (MK79-hemoglobin expressing strain) were exposed to HOCl. In these cells, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), alpha-amylase production, growth and lethality were assessed in the presence and absence of HOCl. It was observed that the hemoglobin makes cells highly susceptible to killing by HOCl. The maximum survival for both strains was with stationary phase cells at any concentration of HOCl. Both strains grown in the presence of 0.0125-0.075 mg/liter HOCl showed a substantial increase in SOD activity and GSH level, with MK79 being the most increased strain in this respect, while the level of CAT activity was decreased in a dose depended manner. Growth of MK57 and MK79 strains decreased as HOCl concentration increased. However, HOCl at concentration above zero enhanced alpha-amylase production (about 2-fold) in both MK79 and MK57. Furthermore, total amylase production (at all HOCl concentrations) by MK79 was always greater than that by MK57. The results indicate that except for survival, the hemoglobin helps cells to grow better and produces more recombinant products and activates general defense systems more in response to oxidative stress when compared with the non-hemoglobin-containing counterpart.     

Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds

α-Amylase levels in intact seeds of barley (Hordeum vulgare L. cv. Himalaya) reach a maximum at 3 to 4 days of germination while gibberellin levels continue to increase beyond 6 days of germination. In contrast to its effect on half seeds, gibberellic acid does not increase the total amount of α-amylase produced in germinating seeds. The inability of gibberellic acid to stimulate α-amylase production is not related to its availability; rather, evidence suggests that a factor(s) in whole seeds prevents further enhancement of α-amylase formation and accumulation. Hydrolysis products accumulate in the subaleurone space of the endosperm of germinating seeds up to concentrations of 570 milliosmolar. Chromatography of these hydrolysis products indicate the presence of maltose and glucose. Calculations based on reducing sugar determinations show that glucose accounts for as much as 57% of the solutes present in the endosperm fluid. Both maltose and glucose in the range of 0.2 to 0.4 M effectively inhibit the production of α-amylase by isolated barley aleurone layers. This inhibition is quantitatively similar to that brought about by solutions of polyethylene glycol and mannitol. On the basis of these data we propose that hydrolysis products which accumulate in the starchy endosperm of germinating seeds function to regulate the production of hydrolytic enzymes by the aleurone layer.     

A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis

Incorporation of starch or casein into protoplast-regeneration medium facilitated shotgun cloning of α-amylase and neutral protease genes from an unidentified Bacillus sp. in Bacillus subtilis by polyethylene glycol-induced protoplast transformation. This modification and the use of the plasmid vector pPL603b enabled us to simultaneously select for promoter-bearing recombinant plasmids that expressed amylase or protease activity. The inserts were found to be 4 and 4.6 kb, respectively. Although protease activity directed by the cloned gene was only 2- to 4-fold higher than for the donor strain, that of α-amylase was 28-fold higher.     

Cloning and Expression of Thermostable α-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable α-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more α-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the α-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the α-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80°C for 60 min.     

pHG276: A multiple cloning site pBR322 copy number vector expressing a functional lacα peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter P_R has been used to direct the synthesis of lacα in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI⁸⁵⁷ gene for temperature regulation of lacα expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lacα and consequently, a Lac⁻ phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.     

Effects of 6-methoxy-2-benzoxazolinone on the germination and α-amylase activity in lettuce seeds

Germination of lettuce seeds was inhibited by 6-methoxy-2-benzoxazolinone (MBOA) at concentrations greater than 0.03 mmol/L. MBOA also inhibited the induction of α-amylase activity in the lettuce seeds at concentrations greater than 0.03 mmol/L. These two concentration–response curves for the germination and α-amylase indicate that the percentage of the germination was positively correlated with the activity of α-amylase in the seeds. Lettuce seeds germinated around 18 h after incubation and inhibition of α-amylase by MBOA occurred within 6 h after seed incubation. These results show that MBOA may inhibit the germination of lettuce seeds by inhibiting the induction of α-amylase activity.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

Nitrite inhibition of Vitreoscilla hemoglobin (VHb) in recombinant E. coli: direct evidence that VHb enhances recombinant protein production

Bacteria engineered with the gene (vgb) encoding Vitreoscilla hemoglobin (VHb) typically produce more protein than unengineered cells, and it has generally been assumed that VHb is responsible for this effect. Here, using matched strains of E. coli that bear a recombinant alpha-amylase gene (MK57) or the alpha-amylase gene and vgb (MK79), we provide evidence supporting this assumption. Sodium nitrite (which is known to inhibit heme proteins) was tested over a range of concentrations regarding effects on growth, alpha-amylase production, respiration, and VHb function in MK57 and MK79. Nitrite concentrations were identified at which respiration of cell membranes was inhibited only slightly and to approximately equal degrees in both strains, while whole cell respiration was inhibited to a greater extent and about twice as much in MK79 as MK57. This suggests that these concentrations inhibit VHb while having a much smaller effect on cytochrome oxidase. Direct measurements of VHb showed, in fact, that the same nitrite concentrations greatly decreased the levels of active (ferrous) and, to a somewhat lesser extent, total (ferrous plus ferric) VHb in MK79. Finally, these same nitrite concentrations reversed the advantage regarding alpha-amylase production of MK79 over MK57 seen at 0 mM nitrite, linking the presence of active VHb with the increase in alpha-amylase production.     

A high-transformation-efficiency cloning vector for Thermus thermophilus.

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10(8) to 10(9) per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the beta-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.     

Alpha-amylase synthesis in wheat kernels as influenced by the seed coat

The effect of seed coat removal on the synthesis of α-amylase isoenzymes in wheat was investigated. The immature wheat endosperm-aleurone (seed coat and embryo detached) produced considerably less α-amylase activity than immature whole or de-embryonated wheat kernels, when incubated under identical conditions of 18.5 C and 99% humidity, in the presence or absence of gibberellic acid (GA₃). The incubated endosperm-aleurone also exhibited unique α-amylase isoenzyme composition when compared to the isoenzyme compositions of incubated whole and de-embryonated immature and mature wheat kernels both in the presence or absence of GA₃. Subsequent studies indicated that the seed coat may contain factor(s) required for normal α-amylase isoenzyme synthesis.     

A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase‐mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57‐SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85–90%. A twofold increase in plasmid yield was also observed for pUC57‐SGS in comparison to pUC57. pUC57‐SGS displayed greater segregational stability than pUC57‐cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064–2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

Background and Aims

α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone.

Methods

α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties.

Key Results

The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population.

Conclusions

The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.     

Amylase synthesis and stability in crested wheatgrass seeds at low water potentials

Drying of seeds of Agropyron desertorum (Fisch. ex Link) Schult. did not result in breakdown of α-amylase nor impair the ability of seeds to resume its synthesis when moistened again. β-Amylase activity did not change during 5 days of germination at a water potential of 0 atmosphere nor during 40 days of incubation at −40 atmospheres. Seeds synthesized α-amylase at 0, −20, and −40 atmospheres, but not at −60 atmospheres. At 0 and −20 atmospheres, the log of α-amylase activity was linearly related to hastening of germination. But at −40 atmospheres, seeds synthesized α-amylase during a time when there was little hastening of germination. Thus, it appears that other biochemical reactions are less drought-tolerant than synthesis of α-amylase. It is concluded that inhibition of α-amylase synthesis is not a controlling factor in the germination of these seeds at low water potentials.     

Molecular cloning of the N-acetylneuraminate lyase gene in Escherichia coli K-12

Summary The gene for N-acetylneuraminate lyase [N-acetylneuraminate pyruvate-lyase; NPL] of Escherichia coli C600 was cloned onto pBR322 as a 9.8 kilobase HindIII fragment of chromosomal DNA and the hybrid plasmid was designated pMK2. The gene in the hybrid plasmid was subcloned in pBR322 as a 1.2 kilobase HindIII-EcoRI fragment and the resultant hybrid plasmid was designated pMK6. NPL activity level was increased more than 5-fold in the pMK6-bearing strain compared with that of the wild type, when the cells were grown on a medium containing inducer (N-acetylneuraminate: NANA). The transformants harbouring pMK6 also showed higher activity even in the absence of inducer. The NPL produced by pMK6-bearing cells was structurally and immunologically the same as that purified from E. coli C600.