ASSESSMENT OF BACILLUS LICHENIFORMIS α-AMYLASE AS A CANDIDATE ENZYME FOR GENETIC ENGINEERING OF MALTING BARLEY1

Bacillus licheniformis α-amylase, a thermostable starch-degrading enzyme, has been assessed as a candidate enzyme for the genetic transformation of malting barley. The temperature optimum, pH optimum and thermostability of B. licheniformis α-amylase were compared with those of barley α-amylase. The bacterial enzyme has a higher pH optimum (?9), a higher temperature optimum (?90°C) and much higher thermostability at elevated temperatures than the barley enzyme. The specific activity of the bacterial enzyme under conditions of pH and temperature relevant to the brewing process (pH 5.5, 65°C) is ?1.5-fold higher than that of the barley enzyme. Measurements of α-amylase activity during a micro-mash showed that the bacterial enzyme is at least as stable as the barley enzyme under these conditions, and that a level of expression for the bacterial enzyme corresponding to ?0.5% of total malt protein would approximately double the α-amylase activity in the mash. B. licheniformis α-amylase activity was rapidly eliminated by boiling following mashing as would occur during brewing. The combined results suggest that barley expressing the bacterial enzyme may be useful in the brewing process.     

Selection of a versatile Lactobacillus plantarum for wine production and identification and preliminary characterisation of a novel histamine-degrading enzyme

In this study, a desirable and versatile Lactobacillus plantarum strain possessing great ability of L-malic acid consumption, biogenic amine degradation and resistance to wine harsh environment was obtained through successive screenings. Malolactic fermentation (MLF) in contaminated grape wine and cherry wine (supplemented with histamine, tyramine and cadaverine) conducted by this strain was finished within 24 and 18 days, respectively, and the concentration of histamine, tyramine and cadaverine was decreased by over 57% following MLF. The enzyme from L. plantarum responsible for the amine degradation was purified to homogeneity by four steps including cell disruption, ammonium sulphate fractionation, an anion chromatography and a gel filtration chromatography. Such enzyme was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after N-terminal sequence analysis and protein Blast, a tetramer enzyme with a subunit molecular weight of 36 kDa. The optimum values of pH and temperature of this enzyme were at pH 7.5 and 40 °C, and stable between pH 5.5–8.5 and 30–50 °C.     

Purification and Characterization of the Commercialized,Cloned Bacillus megaterium α-Amylase. Part I: Purification and Hydrolytic Properties

An amylolytic enzyme, originally isolated from Bacillus megaterium. was shown to increase the maximum amount of dextrose produced during saccharification by decreasing the amount of residual oligosaccharides. The enzyme, now commercially produced in a genetically engineered strain of Bacillus subtilis, was purified to homogeneity from the commercial product. A combination of gel permeation chromatography in the presence of 1.0 M NaCl and chromatofocusing between pH 9.0 and 7.0 were used to obtain the pure enzyme. The molecular weight of the Bacillus megaterium α-amylase was 59,000 by SDS gel electrophoresis, and the isoelectric point was 8.9 to 9.0. The enzyme was shown to possess both hydrolytic and transferase activity. The enzyme hydrolyzed a wide variety of soluble substrates. The rate of hydrolysis was greatest on soluble starch; α(1,6)-branched substrates and cyclodextrins were hydrolyzed more slowly.     

In Situ De-esterification of lime pectin

The in situ de-esterification of pectin in lime pulp by the action of pectinesterase (PE) has been investigated. It has been shown that the degree of pectin esterification is reduced to about 20% when the pulp is held at pH 8.5 for 90 min. The rate of de-esterification by the enzyme in situ is highest when the pH is in the range 7.5–9.0 and the NaCl concentration is 0.1–0.3M. At pH values above 9 chemical de-esterification becomes important. The activity of extracted lime PE was shown to be almost independent of pH in the range pH 6.0–9.0. It is suggested that the difference between the behaviour of the extracted and the in situ enzyme is due to the fact that the latter needs to be solubilised before it can act on some of the pectin in the pulp. In support of this it is found that the proportion of lime PE which can be extracted from the pulp decreases with decreasing pH and ionic strength, reflecting electrostatic binding to the cell wall.     

Flax retting by degumming composite enzyme produced by Bacillus licheniformis HDYM-04 and effect on fiber properties

Flax enzymatic retting with composite enzyme produced by microbes with inexpensive substrates is widely researched due to less contamination and lower cost. Bacillus licheniformis HDYM-04, isolated from a liquid sample of flax retting pool, efficiently produced degumming enzymes after 48 h of fermentation with inexpensive konjaku flour, consisted of 587.5 U/mL pectinase, 365.2 U/mL mannanase, and 140.1 U/mL xylanase. Almost half the maximum activity of three above-mentioned degumming enzymes was maintained at pH 4.0–6.0 which demonstrated its stability in pH condition of flax retting. After 120 h of retting with this composite enzyme, scanning electronic microscopy showed more significant reduction in gummy components on the fiber surface than those of water retting. The fiber strength was 182.4 ± 9.3 N, 14.3% higher than water-retted samples. The long fiber rate and fiber yield also verified higher fiber productivity. The results permitted this degumming composite enzyme an applicable potential in flax retting.     

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0?7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca ²⁺ , Mn²⁺, PCMB, and DTNB, but inhibited by Ba²⁺, Fe³⁺, Hg²⁺, Mg²⁺, and iodoacetic acid. The purified enzyme was of α?-type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α?-1,4-linkages and with increasing percentage of α?-1,6-linkages in substrate similar to the results from commercially available honey.     

Purification and Properties of Beta-galactosidase from Streptococcus thermophilus

A β-galactosidase from Streptococcus thermophilus was purified to homogeneity by ammonium sulfate and acetone fractionation, gel filtration on Sephadex G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme preparation exhibited an optimum pH at 6.6–7.0 and an optimum temperature of 57°C. The enzyme was stable at pH 6.8–7.0. Km and V_max for the enzyme, using ortho-nitrophenyl β-D-galactopyranoside as the substrate, were 0.25 mM and 83 μmoles/mg protein/min, respectively. It was strongly inhibited by Hg⁺⁺, Ag⁺, and Cu⁺⁺ as well as pchloro-mercuri benzoate. The enzyme had a molecular weight of about 6 × 10⁵ and was highly specific for β-galactoside bonds.     

Purification and Characterization of a Thermostable alpha-Amylase from Bacillus licheniformis

The heat stability of a bacterial α-amylase is important for industrial starch utilization. Although extensive studies have been done on heat stable α-amylase from various bacterial species little is known about the α-amylases of Bacillus licheniformis. In order to get better understanding of thermostable amylases produced by different strains of B. licheniformis and provide information how to utilize the enzyme in starch processing, studies on purification and characterization of a commercial heat stable bacterial α-amylase from B. licheniformis BLM 1777 are reported.     

Functional expression of recombinant sweet-tasting protein brazzein by Escherichia coli and Bacillus licheniformis

Brazzein is an attractive sweetener candidate because of its sugar-like taste, high sweetness, and good stability at high temperature and wide pH range. This study was aimed to express and purify bioactive recombinant brazzein (rBrazzein). The rBrazzein gene was synthesized according to the preferred codons of Bacillus subtilis and successfully expressed in Escherichia coli and Bacillus licheniformis. In E. coli host, lower induction temperature of 30°C increased soluble rBrazzein (Ebrazzein) at high level. In B. licheniformis host, two signal peptides (Sec type and Tat type) were evaluated for the expression of rBarzzein in B. subtilis and B. licheniformis. However, only the Sec-type signal peptide guided the secretion expression of rBrazzein in B. licheniformis. The rBrazzein was expressed steadily and the highest yield reached about 57 mg/L at 36 h by small-scale fermentation. The purification procedure of rBrazzein by B. licheniformis (Bbrazzein) was thus established. Approximately 5 mg/L purified rBrazzein was obtained and the purity was 85%. The conformational state of rBrazzeins was confirmed by circular dichroism. The bioactivities of rBrazzeins were evaluated by sweet taste testing. The Bbrazzein and Ebrazzein were 266 times and 400 times sweeter than sucrose on a weight basis, respectively. The formation of disulfide bonds were both confirmed by LC/MS/MS and MALDI-TOF. The CD analysis indicated that Ebrazzein has a similar secondary structure with natural brazzein, which explained why Ebrazzein had a higher intensity of sweetness. This study demonstrated that B. licheniformis system is useful to produce active recombinant brazzein, and has potential food industry applications.     

A Maltooligosaccharides Producing α-Amylase from Bacillus subtilis SDP1 Isolated from Rhizosphere of Acacia cyanophylla Lindley

Maltooligosaccharides producing amylases are required in the food industry, especially in breadmaking. The Bacillus subtilis strain SDP1 amylase hydrolyses starch to produce maltotriose and maltotetraose along with maltose after prolonged reactions of 5 h. Bacillus subtilis strain SDP1 was isolated from the rhizosphere of Acacia cyanophylla Lindley from the Çukurova region of Turkey. The highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37°C. Under optimized culture conditions, 68.49 U/mL activity was obtained. SDP1 α-amylase had molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and was highly active at pH ranging from 5.0 to 9.0. The optimum temperature of the crude enzyme was 60°C, and it retained 83% and 74% of its initial activity after 1 h and 2 h incubation periods, respectively, at 50°C. While, Mn⁺² has a stimulatory effect on the activity, Ca⁺², Mg⁺², Na⁺ did not effect the enzyme activity. Fe⁺³, Ni⁺², Cu⁺² and Co⁺² had an inhibitory effect on SDP1 amylase activity.     

Determination of the influence of the pH of hydrolysis on enzyme selectivity of Bacillus licheniformis protease towards whey protein isolate

Enzymatic protein hydrolysis is typically described by the degree of hydrolysis and by the enzyme specificity. While the specificity describes which cleavage sites can potentially be cleaved, it does not describe which are preferred. To identify the relative rate at which each individual cleavage site is hydrolysed, enzyme selectivity has recently been introduced. To test the effect of pH on selectivity, whey protein isolate (WPI) was hydrolysed with Bacillus licheniformis protease (BLP) at pH 7.0, 8.0 or 9.0. At all pH values, large differences in the enzyme selectivity (from <0.004% to 27%) towards the different cleavage sites of β-lactoglobulin were observed. The changes in selectivity as a function of pH were significant (up to 80% increase or decrease). This significant variation in selectivity shows that this parameter may be useful in understanding peptide release kinetics in enzymatic hydrolysis under different conditions.     

A novel cyclodextrin glycosyltransferase from an alkalophilic Bacillus species: purification and characterization

Cyclodextrin glycosyltransferase (E.C. 2.4.1.19) of alkalophilic Bacillus sp. 7-12 was purified by ammonium sulfate precipitation, DEAE–cellulose column chromatography and Sepharose CL-6B column chromatography. The enzyme thus obtained consisted of a single band that did not dissociate into subunits by SDS–polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was determined to be 69,000 Da by SDS–PAGE. The enzyme was stable below 70 °C with an optimum activity at 60 °C, and was stable at a pH range of 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by MgCl₂, ZnCl₂, CuSO₄, Al₂(SO₄)₃, CoCl₂, AgNO₃, FeSO₄ and slightly inhibited by SnCl₂ and MnCl₂. CaCl₂, KCl, EDTA and DTT had no influence on the enzyme activity. For cyclodextrin production, up to 34% conversion to cyclodextrins was obtained from 10% starch. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.26:1:0.86.     

EFFECTIVE PURIFICATION PROCEDURE OF ASPERGILLUS ORYZAEα-AMYLASE FROM SOLID STATE FERMENTATION CULTURES INCLUDING CONCANAVALIN A-SEPHAROSE

Solid state fermentation cultures of Aspergillus oryzae NRRL 3485 on moistened wheat bran produced high levels of α-amylase as judged by the specific activity of water extracts (500–700 units/mg protein) and by the enzyme concentration (around 0.15 g/L) in those extracts. The purification of α-amylase by affinity chromatography on Concanavalin A-Sepharose is described. A first DEAE-Sepharose chromatography step (binding of the enzyme contained in the water extract and further elution with 0.2 M NaCl, pH 5) was necessary in order to remove contaminants that hinder the binding of the glycoprotein α-amylase to the lectin. Elution was performed with 7.5 mM α-D-methylmannoside. The enzyme was obtained highly concentrated and purified with a specific activity of 2000 units/mg protein and a recovery of around 60%, free of α-glucosidase, amyloglucosidase and color contaminants. The purity of the enzyme preparation was assessed through nondenaturing gel electrophoresis and sucrose gradient centrifugation. An improvement of the performance of the purification procedure is presented in which the maximum capacities of both the anion exchanger and the lectin matrices were exploited.     

THERMAL AND HIGH-PRESSURE STABILITY OF PURIFIED PECTIN METHYLESTERASE FROM PLUMS (PRUNUS DOMESTICA)

Pectin methylesterase (PME) from greengage plums (Prunus domestica) has been extracted and purified using affinity chromatography. Only one band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis was obtained, with an estimated molecular weight of 31 kDa. On isoelectric focusing electrophoresis, two bands with neutral isoelectric points (6.8 and 7.0) were detected. The optimal pH and temperature for plum PME activity were 7.5 and 65C, respectively. A study of purified plum PME thermostability was performed at pH 7.5 and 4.0, indicating a higher thermostability at pH 7.5 than at pH 4.0. A biphasic inactivation behavior was observed for thermal treatments (54–70C), whereas its pressure inactivation could be described by a first‐order kinetic model in a pressure range of 650–800 MPa at 25C. Purified plum PME was found to be relatively stable to thermal and pressure (≤600 MPa) treatments, compared to PME from other fruits.     

The Use of Red Oncom Powder as Potential Production Media for Fibrinogenolytic Protease Derived from Bacillus Licheniformis RO3

The high cost of enzyme production is one of the barriers to successful application of enzyme in the industry. Selection of media is a critical factor for the enzyme production. Main factors for optimization of enzyme production include nutritional components and environmental conditions for growth and production of fibrinogenolytic protease. In this study, Bacillus licheniformis RO3 isolated from red oncom, an Indonesian fermented food was tested for its fibrinogenolytic protease production by using several media. Three types of media were analyzed, i.e. Luria-Bertani broth (LB), ½ LB + 1% skim milk (LBS), and ½ LB + 1% red oncom powder (LBO). Protease activity was tested by using spectrophotometric method with casein as a substrate and fibrinogenolytic activity was confirmed based on zymography assay using fibrinogen substrate. In LB media, B. licheniformis RO3 was able to produce protease with activity of 0.024 U/ml or 0.157 U/mg at 36 h fermentation. In LBS media, the highest protease activity was 0.022 U/ml or 0.152 U/mg at 48 h fermentation. The best result was shown by B. licheniformis RO3 grown in LBO media with the highest protease activity of 0.051 U/ml or 0.283 U/mg at 48 h. Zymographic profiles showed that crude enzyme from B. licheniformis RO3 consisted of six fibrinogenolytic bands with molecular weight of 20, 27, 32, 40, 70, and >140 kDa. These results indicate that red oncom powder can be used as a potential media for fibrinogenolytic protease production.     

Microbial α-Galactosidase for Soymilk Processing

Several microorganisms isolated from soil enriched with soybean meal were screened for their ability to produce α-galactosidase (α-D-galactoside galactohydrolase, E.C. 3.2.1.22). Soybean carbohydrates, after fermentation with Saccharomyces cerevisiae, were used as carbon source for an effective screening. Fructose-free soy oligosaccharides were effective as inducers of the enzyme whereas normal soy oligosaccharides, including sucrose, were not. With this carbon source, invertase production was insignificant and the enzymes present liberated galactose from oligosaccharides known to be present in soybeans. Among the microorganisms studied, Cladosporium cladosporioides (Fres.) de Vr. had the highest α-galactosidase activity. The intracellular enzyme had an optimum pH around 7.0 on the artificial substrate p-nitrophenyl-α-D-galactopyranoside (PNPG) but a pH optimum of 5.0 with melibiose. At 6.3 (the natural pH of soymilk) the relative activity on the two substrates was 90%. The enzyme was relatively thermostable, with no detectable decrease in activity when held for more than 16 hr at 47°C or 2 hr at 60°C. The enzyme liberated galactose from melibiose, raffinose, and stachyose and eliminated raffinose-like sugars from soymilk, specially stachyose, in a few hours.     

Characterization and Cheese-Making Properties of Rennet-Like Enzyme Produced by a Local Algerian Isolate of Aspergillus niger

An ochratoxin free extracellular acid protease was produced by solid state cultivation of Aspergillus niger FFB1. The purified enzyme (48.7 kDa) showed an optimal milk clotting activity at pH 5.5 and 45°C in the presence of 0.01 M CaCl₂. The enzyme was stable at least 24 h at 35°C in the pH range of 5.5–7.0. Thermal denaturation started above 45°C. Fresh cheese manufactured with reconstituted cow milk and the purified enzyme showed similar basic characteristics (pH 4.5, acid taste, white color) as marketed cheeses obtained with calf rennet. This emphasizes the value of exploiting local biological resources for value added food processing in developing countries.     

Effect of pH on the gel properties and secondary structure of fish myosin

The relationships between gel properties and the secondary structures of silver carp myosin were investigated at pH 5.5–9.0 using dynamic rheological measurement, circular dichroism and scanning electron microscopy. The gel properties of fish myosin were strongly pH and temperature dependent. During heating at 1 °C/min, myosin formed gels in the pH range 5.5–7.5, but not at pH 8.0–9.0. α-Helix was the predominant structure at pH 7.0. The α-helix fraction declined with increasing temperature and the pH away from 7.0, whilst the other secondary structure fractions increased. The α-helix structure of myosin was more susceptive to acid-treatment than alkali-treatment. As pH increased, the gelation rate and gel strength decreased, and the water-holding capacity (WHC) showed an increasing trend followed by a plateau. High β-sheet and β-turn fractions prior to heating could improve G′ at 90 °C, but they depressed the WHC. A compact and uniform gel of fish myosin was obtained at pH 7.0.     

Production and characterization of partially purified thermostable α-galactosidases from Streptomyces griseoloalbus for food industrial applications

Solid-state fermentation was carried out for the production of extracellular α-galactosidase by Streptomyces griseoloalbus. Soybean flour was the best solid substrate for α-galactosidase production. In flask-level optimization, the highest enzyme yield of 111 ± 0.2 U/gds was obtained under optimal conditions. The partially purified α-galactosidase preparation showed highest activity at pH 5.0 and 65 °C. The enzyme was completely stable at pH 5.0 to 7.0 and at 50 and 55 °C for 5 h. The t_1/2 of the enzyme at 65 °C was 3.5 h. The information obtained from the present investigation is advantageous for food industrial applications of S. griseoloalbus α-galactosidases.     

Purification and Characterization of a Pullulan-Hydrolyzing Glucoamylase from Sclerotium rolfsii

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50°C between pH 4.0–4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0–4.5 and at 65°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml⁻¹ (V_max 1.0U · mg⁻¹) and for soluble starch, it was 0.6mg · ml⁻¹ (V_max 8.33 U · mg⁻¹). The enzyme was observed to be a glycoprotein (12–13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed α-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 μM). The enzyme has been characterized as a glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.