Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

一株嗜热地衣芽孢杆菌及其产高温淀粉酶的初步研究

从云南腾冲热海热泉中分离到23株产高温淀粉酶的菌株,选取酶活最高的一株菌株进行生长特征、16S rRNA基因测序及系统进化分析表明,该菌株为嗜热的地衣芽孢杆菌(Bacillus licheniformis),并命名为B.1icheniformis Tamy6。该菌株生长范围为37～70℃,最适生长温度为55℃。对该菌所产高温淀粉酶的性质研究表明:该酶在70℃具有最高催化效率,在98℃保温30min,仍有45%的活力,其最适反应pH为5.0。通过Native-PAGE酶谱分析表明菌株Tamy6的粗酶液中含有一种类型的淀粉酶。通过TLC分析水解淀粉产物表明,其产物主要为葡萄糖、麦芽糖及3～5个葡萄糖基的寡糖,说明菌株Tamy6所产淀粉酶为高温α-淀粉酶。     

Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u

Four kinds of thermostable chitinase were isolated from the cell-free culture broth of Bacillus licheniformis X-7u by successive column chromatographies on Butyl-Toyopearl, Q-Sepharose, and Sephacryl S-200. We named the enzymes chitinases I(89 kDa), II(76 kDa), III(66 kDa) and IV(59 kDa). Chitinases II, III and IV possessed extremely high optimum temperatures (70-80 degrees C), showing remarkable heat stability. Chitinases II, III and IV produced (GlcNAc)2 and GlcNAc from colloidal chitin and chitinase I predominantly produced (GlcNAc)2. The action pattern of chitinase I on PN-(GlcNAc)4 also showed a stronger propensity to cleave off the (GlcNAc)2 unit from the non-reducing end than the other three chitinases. Chitinases II, III and IV catalyzed a transglycosylation reaction that converted (GlcNAc)4 into (GlcNAc)6.     

Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45 degrees C and the half-life was 1 h at 64 degrees C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chain fatty acid esters. The enzyme had a KM of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37 degrees C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Genotypic diversity among Bacillus licheniformis strains from various sources

Bacillus licheniformis is exploited industrially for the production of enzymes and has been shown to exhibit pathogenic properties. Because of these divergent characteristics, questions arise concerning intraspecies diversity. A comparative study by means of combined repetitive polymerase chain reaction, rpoB and gyrA sequencing, 16S rDNA targeted probe analysis, DNA-DNA hybridizations, gelatinase tests and antibiotic susceptibility tests was performed on a set of strains from diverse sources, including strains with pathogenic potential. B. licheniformis was found to consist of two lineages that are distinguished genotypically.     

Potential application of two thermostable lichenases from a newly isolated Bacillus licheniformis UEB CF: Purification and characterization

Two thermostable and alkali-stable β-1,3–1,4 glucanases (EC 3.2.1.73) EG1 and EG2 from a newly isolated Bacillus licheniformis UEB CF were purified. The molecular weights of EG1 and EG2 enzymes determined by SDS–PAGE were approximately 30 kDa and 55 kDa, respectively. The N-terminal amino acid sequences of EG1 and EG2 β-glucanases were determined to be GAAPIKKGTTKLN and DINGGGATLPQK, respectively. The optimum temperature, optimum pH, k_m and V_max of EG1 were 70 °C, 5.0, 2.1 mg/ml and 21.25 μmol/min/mg, respectively. These values for EG2 were 60 °C, 7.0, 1.82 mg/ml and 18.54 μmol/min/mg, respectively.Both endoglucanases were highly active against barley β-glucan and lichenan. However, they were inactive against CMC and laminarin. The purified β-glucanases were found to be relatively stable toward non-ionic surfactants and oxidizing agents. In addition, both enzymes showed excellent stability and compatibility with a wide range of commercial solid detergents suggesting that they are a potential candidate in detergent industries formulation.     

General characteristics of thermostable amylopullulanases and amylases from the alkalophilic Bacillus sp. TS-23

An alkalophilic strain of Bacillus sp., designated TS-23, was isolated from a soil sample collected at a hot spring (Tainan, Taiwan). During growth in a medium containing 1% soluble starch as the sole source of carbon, the fermentation broth exhibited both pullulanase and amylase activity. Pullulanase and amylase activities were maximal at 65° C. The pH optima were 8.8 to 9.6 for pullulanase and 7.5 to 9.4 for amylase. Under optimal conditions, a crude preparation hydrolysed pullulan, generating maltotriose as the major product. Strain TS-23 was found to produce five amylases (A_c, A₁, A₂, AP₁, and AP₂), which were visualized by activity staining of proteins that had been separated by native polyacrylamide gel electrophoresis. Both AP₁ and AP₂ had pullulanase activity and A_c, A₁ and A₂ had the ability to adsorb to raw corn-starch. Native corn-starch was partially digested by adsorbed amylases during the course of 12 h at 50° C, with initiation of granular pitting. Further incubation of the reaction mixture resulted in considerable morphological changes in corn-starch granules, and the main soluble products were maltose, maltotriose and higher oligosaccharides.     

Characterization of a thermostable alkaline phytase from Bacillus licheniformis ZJ-6 in Pichia pastoris

A novel neutral phytase gene (phyC) from Bacillus licheniformis was cloned and expressed in Pichia pastoris under the control of AOX1 promoter. The gene is 1,146 bp in size and encodes a polypeptide of 381 amino acids. The recombinant PhyCm (rePhyCm), driven by the Saccharomyces cerevisiae α-mating factor, was secreted into culture medium. After 0.5% methanol induction for 96 h, the activity of rePhyCm in culture supernatant reached 0.23 U/ml. The optimum temperature and pH of purified rePhyCm were 60°C and 7.5, respectively. The rePhyCm was stable in a wide pH range of 5.0–9.0, especially for alkaline pH. The residual activities of rePhyCm retained over 80% after being incubated at pH 5.0–9.0, 37°C for 1 h in the presence of 1 mM CaCl₂. Interestingly, supplemental Ca²⁺ upgraded both the thermostability and pH stability of rePhyCm. Substrate specificity of rePhyCm, effects of metal ions and chemicals on phytase activity were also investigated in current study.     

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.     

Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.     

Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM 13/ATCC14580) was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. These were selected by using a consensus approach together with assessing protein stability changes (PoPMuSiC) and B-factor iterative test (B-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 °C and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing the wild-type RGI lyase in Bacillus subtilis as opposed to in Pichia pastoris; this effect is suggested to be a negative result of glycosylation of the P. pastoris expressed enzyme. A ~ twofold improvement in thermal stability at 60 °C, accompanied by less significant increases in T _m of the enzyme mutants, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects.     

Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations

The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0–11.0 and 65–70 °C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 °C.

The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 °C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 °C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.     

Purification and properties of penicillinases from two strains of Bacillus licheniformis: a chemical, physicochemical and physiological comparison

1. The penicillinases formed by penicillinase-constitutive mutant strains from two closely related varieties (749 and 6346) of Bacillus licheniformis have been isolated, characterized and compared. They are chemically, physicochemically and immunologically very similar, but differ enzymologically in absolute and relative activity on, and affinity for, different penicillins and cephalosporins. 2. The molecular weights of both types are approx. 23000. Neither enzyme contains any cyst(e)ine. However, in most other respects they show little resemblance to any of the other penicillinases so far isolated. 3. Their properties, whether isolated from cells (to which approx. 50% of the activity is normally bound) or from the culture supernatant, appear to be similar. However, the molecular weight of a preparation of enzyme from strain 749/C obtained from the culture supernatant was found to be significantly (over 20%) higher than that obtained from cells alone. 4. With benzylpenicillin, the enzyme from strain 749 has V_max. approx. 6 times higher than that of the enzyme from strain 6346, but this difference is `compensated' by its affinity being 6 times lower. Thus, at the very low biologically effective concentrations of penicillin met with under natural conditions, where neither type of enzyme is more than a fraction saturated with its substrate, the antibiotic is hydrolysed at the same rate by both. As expected, the penicillin-sensitivities of single cells from the two strains were found to be identical. 5. It is suggested that the concept of `physiological efficiency' (defined as V_max. divided by K_m), applied to enzymes acting naturally under conditions of poor saturation with their substrates, may be useful for expressing their biological function in vivo.     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.     

High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system

Background  

Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable.     

Cloning of a thermostable α-amylase gene from Bacillus licheniformis and its expression in Escherichia coli and Bacillus subtilis

Abstract The gene coding for the thermostable α-amylase Bacillus licheniformis has been isolated from a direct shotgun in Escherichia coli using the bacteriophage lambda as a vector. The fragment containing the α-amylase gene has been sub-cloned in pBR322 and its restriction map determined. The α-amylase produced by the E. coli clones retained the thermostability of the B. licheniformis enzyme. Expression and properties of the gene product in E. coli and Bacillus subtilis have been examined.     

Chitobiose production by using a novel thermostable chitinase from Bacillus licheniformis strain JS isolated from a mushroom bed

The thermophilic Bacillus licheniformis strain JS was isolated from a bed of mushrooms, Pleurotus sajor-caju. The organism could produce a novel, single-component, thermostable chitinase that was purified by ion-exchange chromatography using DEAE-cellulose in 7.64% yield and in an 8.1-fold enhancement in purity. Its molecular weight is 22 kDa. The enzyme is a chitobiosidase, since the chitin hydrolysate is N^I,N^II-diacetylchitobiose. The optimum temperature for enzyme activity is 55 °C, and the optimum pH is 8.0. It was completely inhibited by Hg²⁺ ions whereas Co²⁺ ions served as an activator. The thermostability of this enzyme is important in the bioconversion of chitinous waste and for the production of chitooligosaccharides.     

Genome sequences of two thermophilic Bacillus licheniformis strains, efficient producers of platform chemical 2,3-butanediol

Both Bacillus licheniformis strains 10-1-A and 5-2-D are efficient producers of 2,3-butanediol. Here we present 4.3-Mb and 4.2-Mb assemblies of their genomes. The key genes for the regulation and metabolism of 2,3-butanediol production were annotated, which may provide further insights into the molecular mechanism for the production of 2,3-butanediol with high yield and productivity.