Extracellular release of recombinant alpha-amylase from Escherichia coli using pulsed electric field

It is difficult for Escherichia coli to secrete products such as recombinant enzymes, because the Gram-negative bacterium has a double membrane structure and so some of the products are accumulated in a periplasmic space. In this study, we demonstrated that recombinant alpha-amylase can be released from recombinant E. coli HB101/pHI301A during cultivation by applying a pulsed electric field (PEF). When a PEF (12 kV, 2 Hz) was applied for 30 min with an interval of 30 min from the point of OD660=0.7, the amount of released alpha-amylase was about 30% of the total amount of alpha-amylase produced in the cells. As a result of SDS-PAGE and activity staining analyses, it was confirmed that the released proteins were not all of the intracellular proteins, and the alpha-amylase, which was identical with intracellular alpha-amylase, was released by applied PEF cultivation. PEF treatment could be useful for easy release of periplasmic protein with selectivity.     

Pulsed electric fields cause sublethal injury in Escherichia coli

AIMS: The objective was to investigate the occurrence of sublethal injury in Escherichia coli by pulsed electric fields (PEF) at different pH values. METHODS AND RESULTS: The occurrence of sublethal injury in PEF-treated E. coli cells depended on the pH of the treatment medium. Whereas a slight sublethal injury was detected at pH 7, 99.95% of survivors were injured when cells were treated at pH 4 for 400 micros at 19 kV. The PEF-injured cells were progressively inactivated by a subsequent holding at pH 4. CONCLUSIONS: PEF cause sublethal injury in E. coli. The measurement of sublethal injury using a selective medium plating technique allowed prediction of the number of cells that would be inactivated by subsequent storage in acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could be useful for improving food preservation by PEF technology and contributes to the knowledge of the mechanism of microbial inactivation by PEF.     

Cloning and expression of raw-starch-digesting alpha-amylase gene from Bacillus circulans F-2 in Escherichia coli

The raw potato-starch-digesting alpha-amylase gene of Bacillus circulans F-2 was cloned for the first time in Escherichia coli C600, using plasmid pYEJ001. The recombinant plasmid, named pYKA3, has a 5.4 kb insert from a chromosome of the donor bacterium. Subcloning of this amylase gene gave plasmid pHA300 which carried 3.15 kb of the inserted DNA. The transformed bacterium, E. coli C600 (pYKA3), produced the amylase in the periplasmic space, whereas it is secreted outside the cell in the donor bacterium. The cloned raw-starch-digesting alpha-amylase has a molecular weight of 93,000 on SDS-PAGE, and its action pattern was absolutely the same as that of the potent raw-starch-digestible amylase produced by B. circulans F-2. The periplasmic amylase produced by the transformed E. coli (pHA300) could digest raw starch granules such as potato, corn and barley raw starch granules, indicating that the raw-starch-digesting amylase is active in E. coli. Furthermore, this amylase crossreacted with the rabbit antiserum raised against the raw potato-digesting alpha-amylase of B. circulans F-2. From these results it was concluded that the cloned amylase is the same amylase protein as B. circulans F-2 amylase, which has a potent raw-starch digestibility. Thus, this paper is to our knowledge the first describing the molecular cloning of raw-starch-digesting alpha-amylase from Bacillus species and its successful expression in E. coli.     

Growth defects of Escherichia coli cells which contain the gene of an alpha-amylase from Bacillus coagulans on a multicopy plasmid

An alpha-amylase gene from Bacillus coagulans has previously been cloned in Escherichia coli and shown to direct the synthesis of an enzymically active protein of 60,000 Dal (Cornelis et al., 1982). In one particular E. coli host, strain HB101, amylase was found to accumulate in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into E. coli 188 which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for beta-galactosidase, a cytoplasmic marker. Abnormally large amounts of both alpha-amylase and beta-galactosidase were found in the culture fluid of cells grown in rich medium. Furthermore a severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by two types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. These results suggest that the synthesis and export of B. coagulans alpha-amylase is deleterious to E. coli, especially in media containing maltose or glycerol as sole carbon source.     

Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.     

Escherichia coli produces a cytoplasmic alpha-amylase, AmyA.

In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the alpha-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase. The newly discovered gene has been named amyA. The natural physiological role of the AmyA protein is not yet evident.     

Secretion activities of Bacillus subtilis alpha-amylase signal peptides of different lengths in Escherichia coli cells

The B. subtilis alpha-amylase promoter and signal peptide are functional in E. coli cells. DNA fragments coding for signal peptides with different lengths (28, 31, 33 and 41 amino acids from the translation initiator Met) were prepared and fused with the E. coli beta-lactamase structural gene. In B. subtilis cells, the sequences of 31, 33 and 41 amino acids were able to secrete beta-lactamase into the surrounding media, but the 28 amino acid sequence was not. In contrast, all of the four sequences were able to export beta-lactamase into the periplasmic space of E. coli cells. Thus, the recognition of the B. subtilis alpha-amylase signal peptide in E. coli cells seems to be different from that in B. subtilis cells.     

Release of periplasmic penicillin amidase from Escherichia coli by chloroform shock

Penicillin amidase is a periplasmic enzyme in Escherichia coli. Conventionally, the periplasmic enzymes are released into the medium by osmotic shock which is tedious involving a number of centrifugation steps. The present communication deals with a simple technique for the release of penicillin amidase by chloroform shock. Experimental findings show that the periplasmic penicillin amidase does not show any variation by the chloroform treatment. This analysis was also extended to the E. coli cells grown at various concentrations of phenylacetic acid, optimal concentration of phenylacetic acid plus glucose and lactic acid.     

Protein release in recombinant Escherichia coli using bacteriocin release protein.

The effect of induction level of the bacteriocin release protein (BRP) on cell growth characteristics, protein expression, and protein release in a recombinant strain of Escherichia coli RR1 was investigated. Mitomycin C, the inducing agent, when added to the growth medium in moderate amounts (up to 200 ng/mL) was observed to enhance the release of periplasmic proteins from the cell to the fermentation broth substantially. The percentages of release of the proteins alpha-amylase and beta-lactamase were increased by factors of about 7 and 3, respectively, upon induction of the BRP. The percentage of alpha-amylase released into the broth increased from only about 5% to almost 50% with the aid of BRP. The cell growth curve and low extracellular activity of the cytoplasmic protein beta-galactosidase were indicative that cell lysis did not occur in an appreciable amount at a low induction level, with a mitomycin C concentration of less than 300 ng/mL.     

Construction of a novel shuttle vector based on an RCR-plasmid from a haloalkaliphilic archaeon and transformation into other haloarchaea

The pNB101 is the first plasmid to be isolated from an haloalkaliphilic archaea. With insertion of the ColE1 replicon of Escherichia coli, as well as two antibiotic resistance genes at its unique Hin dIII site, a novel shuttle vector between haloarchaea and E. coli was developed. This vector, named pNB102, was successfully transformed into two non-alkaliphilic haloarchaea, Halobacterium salinarum SNOB and Haloarcula hispanica ATCC33960. The presence and stability of pNB102 in the transformants were confirmed by PCR identification, Southern blotting and restriction endonuclease digestion. Results also indicated that the presence of restriction-modification (R-M) systems in some Halobacterium species prevented this transformation. It is the first report that the replicon of pNB101 has such a wide host range, and has taken the first step for construction of the vector/host system in haloalkaliphilic archaea.     

Release of outer membrane fragments from wild-type Escherichia coli and from several E. coli lipopolysaccharide mutants by EDTA and heat shock treatments.

EDTA-induced outer membrane losses from whole cells of wild-type Escherichia coli (O111:B4) and several lipopolysaccharide (LPS) mutants derived from E. coli K-12 D21 were analyzed. EDTA treatment induced losses of LPS (up to 40%), outer membrane proteins OmpA, OmpF/C, and lipoprotein, periplasmic proteins, and phosphatidylethanolamine. The extent of these releases was strain specific. Successively more EDTA was necessary to induce these losses from strains containing LPS with increasing polysaccharide chain length. An additional heat shock immediately following the EDTA treatment had no effect on LPS release, but it decreased the release of outer membrane proteins and reduced the leakage of periplasmic proteins, suggesting that the temporary increase in outer membrane "permeability" caused by Ca2+-EDTA treatment was rapidly reversed by the redistribution of outer membrane components, a process which is favored by a mild heat shock. The fact that the material released from E. coli C600 showed a constant ratio of lipoprotein, OmpA, and phosphatidylethanolamine at all EDTA concentrations tested suggests that the material is lost as specific outer membrane patches. The envelope alterations caused by EDTA did not result in cell lysis.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Extracellular production of cloned alpha-amylase by Escherichia coli

Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.     

Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric fields

Two pathogenic microorganisms Escherichia coli O157:H7 and Staphylococcus aureus, suspended in peptone solution (0.1% w/v) were treated with 12, 14, 16 and 20 kV/cm electric field strengths with different pulse numbers up to 60 pulses. Pulsed electric field (PEF) treatment at 20 kV/cm with 60 pulses provided nearly 2 log reduction in viable cell counts of E. coli O157:H7 and S. aureus. S. aureus cells were slightly more resistant than E.coli O157:H7 cells. The results related to the effect of initial cell concentration of E. coli O157:H7 on the PEF inactivation showed that more inactivation was obtained by decreasing initial cell concentration. Any possible injury by PEF was also investigated after applying 20 kV/cm electric field to the microorganisms. As a result, it was determined that there was 35.92 to 43.36% injury in E. coli O157:H7 cells, and 17.26 to 30.86% injury in S. aureus cells depending on pulse number. The inactivation results were also described by a kinetic model.     

Protein secretion controlled by a synthetic gene in Escherichia coli

The inability of Escherichia coli to secrete proteins in growth medium is one of the major drawbacks in its use in genetic engineering. A synthetic gene, homologous to the one coding for the kil peptide of pColE1, was made and cloned under the control of the lac promoter, in order to obtain the inducible secretion of homologous or heterologous proteins by E. coli. The efficiency of this synthetic gene to promote secretion was assayed by analysing the production and secretion of two proteins, the R-TEM1 beta-lactamase, and the alpha-amylase from Bacillus licheniformis. This latter protein was expressed in E. coli from its gene either on the same plasmid as the kil gene or on a different plasmid. The primary effect of the induction of the kil gene is the overproduction of the secreted proteins. When expressed at a high level, the kil gene promotes the overproduction of all periplasmic proteins and the total secretion in the culture medium of both the beta-lactamase or the alpha-amylase. This secretion is semi-selective for most periplasmic proteins are not secreted. The kil peptide induces the secretion of homologous or heterologous proteins in two steps, first acting on the cytoplasmic membrane, then permeabilizing the outer membrane. This system, which is now being assayed at the fermentor scale, is the first example of using a synthetic gene to engineer a new property into a bacterial strain.     

Numerical evaluation of lactoperoxidase inactivation during continuous pulsed electric field processing

A computational fluid dynamics (CFD) model describing the flow, electric field and temperature distribution of a laboratory‐scale pulsed electric field (PEF) treatment chamber with co‐field electrode configuration was developed. The predicted temperature increase was validated by means of integral temperature studies using thermocouples at the outlet of each flow cell for grape juice and salt solutions. Simulations of PEF treatments revealed intensity peaks of the electric field and laminar flow conditions in the treatment chamber causing local temperature hot spots near the chamber walls. Furthermore, thermal inactivation kinetics of lactoperoxidase (LPO) dissolved in simulated milk ultrafiltrate were determined with a glass capillary method at temperatures ranging from 65 to 80°C. Temperature dependence of first order inactivation rate constants was accurately described by the Arrhenius equation yielding an activation energy of 597.1 kJ mol^?1. The thermal impact of different PEF processes on LPO activity was estimated by coupling the derived Arrhenius model with the CFD model and the predicted enzyme inactivation was compared to experimental measurements. Results indicated that LPO inactivation during combined PEF/thermal treatments was largely due to thermal effects, but 5–12% enzyme inactivation may be related to other electro‐chemical effects occurring during PEF treatments. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Emission of carbonyl sulfide by Thiobacillus thioparus grown with thiocyanate in pure and mixed cultures

Abstract The biological activity of the heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was found to be predominantly associated with the periplasmic extract (about four-fold higher than the culture supernatant) of a recombinant E. coli (JM109) strain carrying the NAG-St toxin gene. Four molecular species of NAG-ST, two each from the periplasmic extract and culture supernatant of JM109, were purified. Amino acid sequence analysis of the four NAG-ST peptides isolated by HPLC revealed that they all differed from that of the mature 17-amino acid residue NAG-ST released by V. cholerae non-O1. The M _r-values of the peptides obtained from the periplasmic extract were 4331 and 2785, while those recovered from the culture supernatant were 3154 and 2785. It thus appears that V. cholerae NAG-ST is synthesized as larger molecules in the recombinant E. coli strain. The differences in sizes of the exported NAG-ST molecule could relate to difference in the enzyme cleavage system between E. coli and V. cholerea .     

芝田硫化叶菌新型α-淀粉酶基因在大肠杆菌的克隆和表达

A novel α-amylase gene was amplified from Sulfolobus shibatae by using PCR technique.The amplified 1.7kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM. The novel α-amylase gene in pSBAM was expressed in E. coli. The production of the novel α-amylase activity reached over 8 units/100mL of the culture. The molecular weight of this enzyme was about 61kD by SDS-PAGE. The expressed novel α-amylase protein in E.coli DHSα accounted for about 20 % of the total protein in the recombinant cell. The cooperative action of the novel α-amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.     

Cloning and expression of an alpha-amylase gene from Streptomyces thermoviolaceus CUB74 in Escherichia coli JM107 and S. lividans TK24

A gene coding for a thermostable extracellular alpha-amylase, carried by a 5.7 kb BamHI chromosomal DNA fragment isolated from Streptomyces thermoviolaceus strain CUB74, was cloned into Escherichia coli JM107 using, as a cloning vector, the high-copy-number plasmid pUC8. E. coli containing a recombinant plasmid pQR300 expressed the amylase gene and exported the enzyme into the periplasmic space and the culture medium. The amylase protein expressed by E. coli had the same molecular mass (50 kDa) as that expressed by the Streptomyces parent strain, which suggests that the enzyme is processed similarly by both strains. The amylase gene was also cloned into Streptomyces lividans TK24 using pIJ702 as vector. The enzyme was stable at 70 degrees C when CaCl2 was present.