Optimization and enhanced production of α-amylase and protease by a newly isolated Bacillus licheniformis ZB-05 under solid-state fermentation

Eight different agro-residues were tested for α-amylase and protease production by using Bacillus licheniformis ZB-05. Among them, rice husk (RH) was proved as the best substrate for two enzymes (α-amylase 443 U/g and protease 469,000 U/g). Maximum enzyme production was observed to be 30 % initial moisture, with a growth period of 36 h in 20 and 30 % inoculum volumes for α-amylase and protease, respectively. The best enzyme recovery from solid mass was obtained when extracted with tap water. Among the tested various nitrogen sources, 1 % ammonium sulphate followed by 2 % Bacto liver, 2 % ammonium sulphate and 1 % Bacto casaminoacid served as the best inorganic and organic nitrogen sources for α-amylase and protease production, respectively. As additional carbon sources, 2 % soluble starch enhanced α-amylase production, while 1 % maltose enhanced protease production.     

Predominant growth of α-amylase regulation mutant in continuous culture of Bacillus caldolyticus

The production of α-amylase in batch and continuous cultures of the strain SP of Bacillus caldolyticus was studied using a maltose-casitone medium. The three quantitative parameters of α-amylase production (maximum values of the specific production rate, volumetric productivity and concentration of α-amylase) increased in continuous culture by 2.5, 4.6 and 3.8 times respectively in comparison with a batch culture. It was found that a mutant strain M1 of α-amylase production was predominant in every run of the continuous cultures. The strain M1 differs from the strain SP in that it can produce α-amylase constitutively in a batch culture while the cells of strain SP require maltose for α-amylase production. On the contrary, α-amylase production by the strain M1 was repressed partly by maltose. Moreover, glucose repression on α-amylase production was not observed for the M1 strain while it was remarkable for the strain SP. The above-mentioned properties of the mutant M1 concerning the regulation of α-amylase production are not only advantageous for industrial use but also interesting from the viewpoint of basic microbiology.     

Enhanced extracellular production of α-amylase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by optimization of regulatory elements and over-expression of PrsA lipoprotein

α-Amylase was used as a heterologous model protein to investigate the effects of promoters, signal peptides and over-expression of an extra-cytoplasmic molecular chaperone, PrsA lipoprotein, on enhancing the secretion of α-amylase in Bacillus subtilis. Four promoters and six signal peptides were compared, successively, and the highest yield of α-amylase was achieved under the promotion mediated by P_AprE, a strong constitutive promoter, and secretion by SP_nprE, a signal peptide from B. subtilis. Moreover, under conditions of overexpressed PrsA lipoprotein, the secretion production and activity of α-amylase increased to 2.5-fold. The performance of the recombinant B. subtilis 1A751PL31 was evaluated with a fed-batch fermentation in a 7.5 l fermentor. Optimization of regulatory elements and over-expression of PrsA lipoprotein had a significant effect on enhancing the production of α-amylase in B. subtilis.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Production of α-amylase in a low cost medium by Bacillus licheniformis TCRDC-B13

A low cost synthetic medium producing large quantities of α-amylase has been developed. Bacillus licheniformis TCRDC-B13 isolated from soil was used for α-amylase production. The α-amylase enzyme of this strain showed excellent stability at high temperatures and over a wide pH range. The low cost medium produced 5 times more enzyme than the high cost synthetic medium (using yeast extract and peptone) in shake flasks. In a 2.6-l fermentor, the enzyme production further doubled.     

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

Production,Purification, and Characterization of α-Amylase from Solventogenic Clostridium sp. BOH3

A solventogenic strain of Clostridium sp. BOH3 produces extracellular α-amylase (7.15 U/mg protein) in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate (1 % w/v) and a small amount of starch (0.1 % w/v), which is essential for the expression of α-amylase. In the presence of α-amylase, BOH3 utilizes starch directly without any pretreatment and produces butanol almost equivalent (～90 %) to the production of butanol from glucose. α-Amylase can be purified from culture supernatant by using one-step weak anion exchange chromatography with a yield of 43 %. In peptide fingerprinting analysis, this enzyme shows homology with α-amylase produced by Clostridium acetobutylicum ATCC824. However, the molecular weight is 54 kDa, which is smaller than α-amylase of ATCC824 (84 kDa). This enzyme has optimum temperature at 45–50 °C and optimum pH at 4.5–5.5. Under this condition, the enzyme activity is 91.32 U/mg protein, and its K _m and V _max values are 1.71?±?0.02 mg/ml and 96.13?±?0.15 μmol/min/mg protein, respectively. Activity of this α-amylase can be enhanced (>1.5 times) by addition of Ca²⁺ and Co²⁺ and its activity can be maintained at an acidic pH (pH 3–5) for about 24 h. These unique characteristics suggest that this enzyme can be used for saccharification of starch for production of biofuel in one pot.     

The alpha-amylases as glycosylases, with wider catalytic capacities than envisioned or explained by their representation as hydrolases

In a study undertaken to illustrate the inadequacy of the familiar concept of carbohydrases as hydrolases, crystalline α-amylases from six different sources, as well as crude salivary amylase, were examined and found to catalyze the synthesis of maltose and maltosaccharides from α-d-glucopyranosyl fluoride, a stereoanalog of α-d-glucopyranose. These syntheses apparently involve initial formation of maltosyl fluoride and higher maltosaccharide 1-fluorides, traces of which were found in digests with certain α-amylases. That the reactions are due to the α-amylases themselves and not to some accompanying enzyme(s) appears certain from the purity and diversity of the preparations; their failure (with one exception) to attack α- or β-maltose; the correspondence of the synthesized products with the known specificity of α-amylases for α-1,4-d-glucosidic linkages (and capacity of different α-amylases to hydrolyze saccharides of different sizes). The “saccharifying” α-amylase of B. sublilis var amylosacchariticus was unique in producing maltosaccharides from both α- and β-maltose (i.e., by α-d-glucosyl transfer). However, the entire group of α-amylases had the capacity to promote α-d-glucosyl transfer from α-d-glucosyl fluoride to C₄-carbinol sites, demonstrating for the first time that the catalytic range of α-amylase extends beyond hydrolysis and its reversal. Indeed, all transferred the glucosyl group of α-d-glycosyl fluoride preferentially to C₄-carbinols rather than water—a finding neither anticipated nor explained by the representation of α-amylases as hydrolases.     

Multi-objective optimization of bioethanol production during cold enzyme starch hydrolysis in very high gravity cassava mash

Cold enzymatic hydrolysis conditions for bioethanol production were optimized using multi-objective optimization. Response surface methodology was used to optimize the effects of α-amylase, glucoamylase, liquefaction temperature and liquefaction time on S. cerevisiae biomass, ethanol concentration and starch utilization ratio. The optimum hydrolysis conditions were: 224 IU/g_starch α-amylase, 694 IU/g_starch glucoamylase, 77 °C and 104 min for biomass; 264 IU/g_starch α-amylase, 392 IU/g_starch glucoamylase, 60 °C and 85 min for ethanol concentration; 214 IU/g_starch α-amylase, 398 IU/g_starch glucoamylase, 79 °C and 117 min for starch utilization ratio. The hydrolysis conditions were subsequently evaluated by multi-objectives optimization utilizing the weighted coefficient methods. The Pareto solutions for biomass (3.655-4.380 × 10⁸ cells/ml), ethanol concentration (15.96-18.25 wt.%) and starch utilization ratio (92.50-94.64%) were obtained. The optimized conditions were shown to be feasible and reliable through verification tests. This kind of multi-objective optimization is of potential importance in industrial bioethanol production.     

Culture conditions for the production of amylolytic enzymes by a thermophilic Clostridium sp.

The growth of a thermophilic Clostridium sp. and the production of α-glucosidase, α-amylase and pullulanase were studied under anaerobic conditions using different carbon and nitrogen sources and varying pH values and temperatures. Growth and enzyme activities were highest with soybean meal as the nitrogen source. The optimum concentration was 2.5% [w/v] for the production of α-amylase as well as pullulanase and 2% [w/v] for α-glucosidase. The best carbon source proved to be soluble starch for α-amylase, and pullulanase and maltose for α-glucosidase. Growth and enzyme production reached their optimum at pH 6.5 to 7.0 and 70°C. Under these conditions, the enzyme activities followed exponential growth with maximum yields of α-glucosidase, α-amylase and pullulanase at 28, 36, and 44 h.     

Isolation of a Bacillus subtilis transformant producing thermostable alpha-amylase by DNA from a thermophilic bacterium

A Bacillus subtilis transformant producing thermostable α-amylase was isolated using DNA from a thermophilic bacterium, Thermophile V2. The extracellular α-amylase did not crossreact with a rabbit antiserum against B. subtilis α-amylase. The structural gene for the thermostable α-amylase was integrated at a different locus from B. subtilis α-amylase. It was linked to pyrA. The transformant was not thermophilic, and its upper temperature for growth was similar to that of the host bacterium.     

Modified α-amylase activity among insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais

Fitness cost is usually associated with insecticide resistance and may be mitigated by increased energy accumulation and mobilization. Preliminary evidence in the maize weevil (Coleoptera: Curculionidae) suggested possible involvement of amylases in such phenomenon. Therefore, α-amylases were purified from an insecticide-susceptible and two insecticide-resistant strains (one with fitness cost [resistant cost strain], and the other without it [resistant no-cost strain]). The main α-amylase of each strain was purified by glycogen precipitation and ion-exchange chromatography (≥70-fold purification, ≤19% yield). Single α-amylase bands with the same molecular mass (53.7 kDa) were revealed for each insect strain. Higher activity was obtained at 35-40 °C and at pH 5.0-7.0 for all of the strains. The α-amylase from the resistant no-cost strain exhibited higher activity towards starch and lower inhibition by acarbose and wheat amylase inhibitors. Opposite results were observed for the α-amylase from the resistant cost strain. Although the α-amylase from the resistant cost strain exhibited higher affinity to starch (i.e., lower K_m), its V_max-value was the lowest among the strains, particularly the resistant no-cost strain. Such results provide support for the hypothesis that enhanced α-amylase activity may be playing a major role in mitigating fitness costs associated with insecticide resistance.     

Enzyme formation and release by isolated barley aleurone layers

Aleurone layers, with testa attached, were prepared from degermed, decorticated barley with the aid of a fungal enzyme preparation. The preparations appeared intact under the scanning electron microscope. By using antibiotics only in an early stage preparations were obtained uncontaminated by micro-organisms and which, when incubated under optimal conditions with gibberellic acid, GA₃, produced near-maximal amounts of α-amylase. The enzyme accumulated in the tissue before it was released into the incubation medium. Daily replacement of the incubation medium, containing GA₃, depressed the quantity of α-amylase produced. α-Amylase was also produced in response to gibberellins GA₁, GA₄ and GA₇ and, to a much lesser extent, helminthosporol and helminthosporic acid. A range of other substances, reported elsewhere to induce α-amylase formation, failed to do so in these trials. At some concentrations, glutamine marginally enhanced the quantity of enzyme formed during prolonged incubations. It is confirmed that α-glucosidase occurs in the aleurone layer and embryo of ungerminated barley, and increases in amount during germination. GA₃ is shown to enhance this increase. When embryos arc burnt, to prevent gibberellin formation, no rise in α-glucosidase levels occurs unless GA₃ is supplied to the grains. As the activity of α-glucosidase and other enzymes have been determined as ‘α-amylase’ by some assay methods, their alterations in activity in response to GA₃ necessitates a re-evaluation of the evidence for de novo) synthesis of α-amylase in aleurone tissue.     

Escherichia coli signal peptidase recognizes and cleaves the signal sequence of α-amylase originating from Bacillus licheniformis

The gene encoding the α-amylase from Bacillus licheniformis was cloned, with and without the native signal sequence, and expressed in Escherichia coli, resulting in the production of the recombinant protein in the cytoplasm as insoluble but enzymatically active aggregates. Expression with a low concentration of the inducer at low temperature resulted in the production of the recombinant protein in soluble form in a significantly higher amount. The protein produced with signal sequence was exported to the extracellular medium, whereas there was no export of the protein produced from the gene without the signal sequence. Similarly, the α-amylase activity in the culture medium increased with time after induction in case of the protein produced with signal sequence. Molecular mass determinations by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing of the purified recombinant α-amylase from the extracellular medium revealed that the native signal peptide was cleaved by E. coli signal peptidase between Ala28 and Ala29. It seems possible that the signal peptide of α-amylase from B. licheniformis can be used for the secretion of other recombinant proteins produced using the E. coli expression system.     

Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

Demonstration and preliminary characterization of α-amylase in the sweetpotato whitefly,Bemisia tabaci (Aleyrodidae: Homoptera)

An α-amylase that hydrolyzes unmodified starch or amylopectin azure was demonstrated in crude and partially purified extracts prepared from whole carcasses of sweetpotato whiteflies (SPW) (Bemisia tabaci Genn.).All nymphal instars and adult SPW, including newly eclosed crawlers that had not yet fed on plant materials, were found to have active α-amylase. α-Amylase activity per mg protein was greatest in 1st instars and decreased with age up to the “pupal” stage, with a very slight increase in activity in adults. However, activity per individual did not differ substantially as a function of age.The α-amylase had an apparent molecular weight of about 70 kDa, an isoelectric point of 6.32 and eluted with about 250 mM NaCl from a strongly basic anion-exchange column.The enzyme activity was inhibited by EDTA and not activated by either NaCl or KNO₃. CaCl₂ strongly enhanced activity.α-Amylase activity was greatest at pH 7.0, but there was considerable activity at pHs above 7.0.The K_m of the α-amylase was 1.47 Mm with p-nitrophenyl α-d-malto-heptaoside as substrate.The presence of an amylolytic enzyme in a phloem-feeding insect is unexpected and raises questions about current assumptions of feeding behavior of this species.     

Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris

The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K_M and V_max values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)₈ barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.     

The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting,recombinant α-amylase from Bacillus licheniformis ATCC 9945a

In this study, a new approach for extracellular production of recombinant α-amylase in Escherichia coli was investigated. A gene encoding a highly efficient raw-starch-digesting α-amylase from Bacillus licheniformis ATCC 9945a was cloned and expressed in E. coli. The gene encoding mature α-amylase was cloned into the pDAss expression vector, and secretion of the gene product was regulated by fusion to the signal peptide of DsbA, a well-characterized E. coli periplasmic protein. E. coli BL21 (DE3) carrying pDAss vector containing amylase gene had approximately 2.5-fold higher volumetric enzyme productivity than the natural system. The recombinant enzyme showed higher efficiency for digesting diverse raw starches when compared with the native enzyme and was similar to commercial α-amylase in its ability to hydrolyze raw starches. The properties of the recombinant enzyme demonstrate the potential of the DsbA signal peptide approach for the secretory production of the fully active, industrially important recombinant enzyme.