Bacillus Alcalophilus Research Articles

Repairing performance and bottleneck analysis of concrete with encapsulated oxygen-dependent microbial Agent: Simulation-Aided characterization

Microbial self-healing concrete has natural advantages because of its environmental harmlessness and compatibility. Appropriate nutrients, microbial composition, and carriers are critical for microbial repairing agents to maximize restoration effectiveness in the harsh environment of concrete. However, little attention has been paid to the evolution of the healing process and the underlying mechanisms of bottlenecks. In this study, the potential of calcium lactate as the main nutrient source for Bacillus alcalophilus (B. alcalophilus), an alkali-resistant spore-producing microorganism, has been demonstrated firstly. The repairing performances and bottlenecks of oxygen-dependent microbial agent were then explored. Considering the harsh environment of concrete, calcium sulphoaluminate (CSA) cement was introduced to form a protective shell through artificial aggregate technology. Simultaneously, to reduce the influence of un-hydrated cement particles and to stabilize the continuous supply of calcium ions, the matrix has a higher maturity through 60 days of hydration. The results show that the encapsulated microbial self-healing agent had good repairing effects on cracks introduced after 60 days of hydration. Moreover, the distribution of repairing products in cracks was observed by X-CT, and the microbial remediation ability based on aerobic decomposing organic compounds was verified by simulation. The bottlenecks, shallow restoration depth and uneven distribution of restoration products, are mainly caused by gas transport and microbial biochemical activity. Finally, increasing the oxygen content in the fracture zone is an effective means to promote carbonate production and fracture filling.

Isolation of biotechnologically important enzyme producing Rhizobacteria from seasonal flower beds

This study aimed to isolate biotechnologically important enzyme like amylase and protease producing rhizobacteria from rhizospheric soil. Soil samples were collected from the rhizospherc of five different flowering plants from Curzon hall, University of Dhaka. A good number of bacteria were isolated. Among them 12 bacterial isolates were selected for detailed study based on their amylolytic and proteolytic activity. Starch hydrolysis ratio (SHR) of the studied isolates ranged in between 2.06 and 4.67, casein hydrolysis ratio (CHR) ranged in between 2.27 and 3.92. All the selected isolateswere Gram positive and rod shaped and provisionally identified as the member of the genus Bacillus with five different species viz. Bacillus alcalophilus, B. subtilis, B. pumilus, B. firmus and B. lentus. Amylase and protease production of the studied bacteria were estimated and it was ranged in between 29.11±9.57 to 35.78 ± 9.05 and 230.27±37.08 to 276.47 ± 55.01 U/ml. The highest amylase and protease producer were Bacillus alcalophilus (Ce104/S9/L) which could produce 35.78 ± 9.05 U/ml and Bacillus subtilis (Ca71/S5/L) could produce 276.47 ± 55.01 U/ml of protease, respectively. Maximum production of both amylase and protease was achieved in 24 h of incubation period at 37°C and pH 7.0. Dhaka Univ. J. Biol. Sci. 32(1): 65-71, 2023 (January)

Characteristics of bio-CaCO3 from microbial bio-mineralization with different bacteria species

Nowadays, MICP (Microbially Induced Calcite Precipitation) technology has broad application prospects in civil engineering, such as sand consolidation, crack healing in cement-based materials, solid waste treatment, etc. Nevertheless, there were no systematic studies on the difference of bio-CaCO3 produced by different microbial strains. In this study, XRD, TEM, SEM, STA and AFM were used to investigate the differences in crystalline form, morphology, size, decomposition temperature, and adhesion force of mineralized products. Those products were produced by Photosynthetic bacteria, Bacillus mucilaginosus, Bacillus alcalophilus, Bacillus pasteurii and chemical pathway. The results showed that all four microbial pathways were more productive than the chemical pathway. The calcite crystals formed by the chemical pathway presented an oblique hexagonal lattice structure, while calcite crystals formed by four microbes presented a spherical or irregular polyhedral structure under the action of organic substrate. The calcite crystals formed by Bacillus pasteurii were smallest in size and those formed by Photosynthetic bacteria were the largest, and their crystalline sizes were smaller than that of calcite produced by chemical pathway. The decomposition temperature and adhesion force of chemical CaCO3 were lower than that of bio-CaCO3, with the highest decomposition temperature (765.80 ℃) and the largest adhesion force (68.91 nN) of calcite induced by Bacillus pasteurii.

Assessment of microbial diversity and enumeration of metal tolerant autochthonous bacteria from tailings of magnesite and bauxite mines

The magnesite and bauxite mines are a major source for some minerals and metals. The tailings of these magnesite and bauxite mines contains massive amount of Mn. (3221.2 ± 6.51 & 7102.8.4 ± 10.24), Cd (40.95 ± 2.12 & 892.86 ± 8.48), Zn (951.08 ± 4.31 &724.12 ± 3.18), and Pb (425.8 ± 5.21 & 812.13 ± 9.19 mg Kg−1) with alkaline and acid pH respectively. In the microbial diversity analysis, the results 8 (Thiobacillus thiooxidans, Leptospirillum ferrooxidans, Acetobacter methanolicus, T. intermedius, Bacillus cereus, Sulfobacillus acidophilus, Methylobacterium sp, and Thiobacillus ferooxidans) and 12 (Serratia marcescens, Metalogenium symbioticum 1, Metallogenium symbioticum 2, Bacillus alcalophilus, Aminobacter sp, Naumaniella neustonica, Staphylococcus aureus 1, Methylbacillus sp, Pandoraea sputorum, Acenetobacter sp, S. aureus 2, Pseudomonas aeruginosa) bacteria species were isolated from bauxite and magnesite mine tailings. The fungus such as Rhizopus arrhizus, Mucor sp, Aspergillus niger and Penicillium sp were obtained in both acid and alkaline environment. Among this microbial diversity, P. sputorum and B. cereus (identified through 16S rDNA sequencing) were shown better resistance to these four metals for up to 250 mg L−1. The bacterial diversity indexes, concludes that there was least diverse among the three sites. The evenness/equitability index also conform lower level of variation among these sites.

Biocontrol of anthracnose on yellow passion fruits by antagonic bacteria to plant pathogens

– In this study was evaluated the efficacy of the bacterial strains: BB-4 ( Bacillus cereus GC, subgroup B), BS-2 ( Photorhabdus luminescens ), BB- 1 ( Bacillus alcalophilus ), BS-5 ( B. cereus GC, subgroup B), BB-5 ( Stenotrophomonas maltophilia ), BB-6 ( Yersinia bercovieri ) and BS-6 ( P. luminescens ) on the control of anthracnose in yellow passion fruit, on antagonism tests and fruits tests under laboratory conditions. The plant pathogen was grown in Petri dishes containing BDA medium for 15 days and the bacterial strains were multiplied in AN medium for two days at 28°C. In passion fruits test, a wound was performed at a depth of 2 mm, followed by spraying with a suspension containing a bacterial antagonist strain (10 8 cfu. mL -1 ). On the wound, a BDA disc with 4 mm in diameter completely colonized by the pathogen and maintenance for 7 days (25oC, high RU% and 12 hours of photoperiod) until the evaluation of diameter of lesions. The experimental design adopted was the completely randomized (9 treatments and 4 replications). The data were submitted to variance analysis and Tukey test (5%). In antagonism test, only BS-5 ( B. cereus GC, Subgroup B) and BS-2 ( P. luminescens ) strains did not inhibit the mycelial growth of the pathogen. The other strains were efficient in inhibition, with levels ranging from 35% to 53%, highlighting the strains BB-4 ( B. cereus GC. Sub group B), BS-3 ( P. luminescens ) and BB-4 ( S. maltophilia ), with 52.5%, 52.5% and 53.3% of control, respectively. In the fruit test, BS-2 ( P. luminescens ), BS-5 ( B. cereus GC, Subgroup B), BB-1 ( B. alcalophilus ), BS-3 ( P. luminescens ) and BB-6 ( Y. bercovieri ) inhibited the development of the pathogen, with control levels varying between 23.4% and 43.6%, with emphasis on BB-1 ( B. alcalophilus ) and BS-3 ( P. luminescens ), with 43.6% of control, indicating potential of use on the biocontrol of anthracnose on passion fruits.

Bacillus alkalilacus sp. nov., isolated from a sediment sample from a lake in India.

An aerobic, endospore-forming, haloalkali-tolerant, Gram-stain-positive, motile, rod-shaped bacterium, designated strain AK73T, was isolated from a sediment sample collected from Sambhar lake, Jaipur, Rajasthan, India. Colonies were circular, 1-2 mm in diameter, glossy, smooth, yellowish and convex with an entire margin after 48 h growth on marine agar at pH 9 and 37 °C. Growth occurred at 15-42 °C, 0-10 % (w/v) NaCl and at a pH range of 7-12. Strain AK73T was positive for catalase and arginine dihydrolase 2 activities, hydrolysis of Tweens 20, 40 and 80, and negative for esculinase, caseinase, gelatinase, β-galactosidase, lipase (Tween 60) and urease activities. The fatty acids were dominated by branched iso-, anteiso-, saturated fatty acids with a high abundance of iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and anteiso-C17 : 0; MK-7 was the major menaquinone. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid, four unidentified phospholipids and three unidentified lipids. The DNA G+C content of strain AK73T was 54 mol%. Analysis based on comparative 16S rRNA gene sequence analysis indicated that Bacillus alcalophilus was the nearest phylogenetic neighbour, with a pair-wise sequence similarity of 96.0 %. Phylogenetic analysis showed that strain AK73T formed a separate lineage but was loosely associated with a peripheral cluster of organisms that contained Bacillus gibsonii, Bacillus murimartini and Bacillus plakortidis with similarity values of 93.6, 93.5 and 93.4 %, respectively. Based on its phenotypic characteristics and on phylogenetic inference, strain AK73T represents a novel species of the genus Bacillus, for which the name Bacillus alkalilacus sp. nov. is proposed. The type strain is AK73T (=JCM 32184T=MTCC 12637T=KCTC 33880T).

Diversity and Characteristics of Kenaf Bast Degumming Microbial Resources

ABSTRACTKenaf is one of the most important natural fiber crops. Traditional degumming with water retting causes serious environmental pollution and reduces the quality of fiber products. The development of kenaf industry is hindered by high production cost. Microbial degumming is suitable for kenaf bast degumming because of its high efficiency, energy conservation, low pollution, and high quality, among others. Through enrichment and screening from water sample, soil sample, and humus sample, we concentrated and identified 92 bacterial strains that could degrade kenaf bast colloid. The strains belonged to 11 genera and 16 species. Five of these strains did not produce cellulase and the weight loss rate of the kenaf bast fiber raw material was more than 20%. These strains belonged to Bacillus subtilis, Paenibacillus polymyxa, Clostridium acetobutylicum, Bacillus alcalophilus, and Erwinia chrysanthemi and were assigned with serial numbers from K1–K5, respectively. This study is the first to report the function of Bacillus pumilus, B. alcalophilus, Clostridium tertium, Brevibacillus brevis, Pectobacterium carotovora, E. chrysanthemi, and Tyromyces subcaesius in kenaf bast degumming. Pectinase and mannanase were the key enzymes in the degumming of kenaf bast.

Properties of Antibiotic-Resistant Bacteria Isolated from Onsite Wastewater Treatment Plant in Relation to Biofilm Formation

The aim of the present study was to determine some properties of antibiotic-resistant bacterial strains isolated from onsite wastewater technology in relation to biofilm formation, e.g., autoaggregation and motility. Additionally, biosurfactant production by the isolates was also evaluated. The ability of selected strains to develop a biofilm was assessed by using the crystal violet method, which allows to indirectly quantify the attached bacterial biomass (live, dead cells, and polysaccharides as well). Obtained results showed that 19 of the analyzed strains were able to produce biofilm after 72 h of incubation. The low values of surface tension in the range between 28 and 36 mN/m were observed in the bacteria, which are not able to produce biofilm or be classified as weak biofilm producers. Among biofilm-forming strains the highest autoaggregation index was observed for Mycobacterium brumae and Bacillus alcalophilus. Noteworthy, that some strains capable of biofilm formation showed no aggregation abilities or were characterized by low autoaggregative properties. The results of visual autoaggregation assay showed no visible flocs after given time of incubation. The results from motility test demonstrated that most of the analyzed strains were motile. Noteworthy, that up to now literature data about physiology, biofilm formation, and autoaggregative capabilities of bacteria isolated from onsite wastewater technology are very limited and this paper gives the information on the antibiotic-resistant bacteria with ability to form biofilm. Thus, the present study points to develop novel bioinocula in antibiotic degradation and to reach novel biofilm-dispersing agents produced by various bacteria that can be used as disinfectants or surface-coating agents to prevent microbial surface colonization and biofilm development.

Structural homogeneity in microbial lipases.

Enzyme lipase (triacylglycerol hydrolases E.C.3.1.1.3) catalyzes the hydrolysis of triacylglycerols to glycerol and free fatty acids. In recent years, progressively more consideration is being rewarded to lipases produced by microorganisms. In nature, some of the most important lipase-producing bacteria are Pseudomonas, Bacillus, Staphylococcus, etc., and fungi are Rhizopus sp., Aspergillus, Penicillium, Geotrichum, Mucor, Rhizomucor, etc. [1,2]. The bacterial lipases from Bacillus sp. exhibit interesting properties that make them potential candidates for biotechnological applications [1-3]. Bacillus alcalophilus are the most common lipase producing strains. Lipases are classified into different families based on the amino-acid sequences similarity and their biological properties [4]. Microbial lipases are relatively stable and are capable of catalyzing a variety of reactions; they hold immense potential for diverse industrial applications.

Paralkalibacillus indicireducens gen., nov., sp. nov., an indigo-reducing obligate alkaliphile isolated from indigo fermentation liquor used for dyeing.

Obligately alkaliphilic, indigo-reducing strains, designated Bps-1T, Bps-2 and Bps-3, were isolated from an indigo fermentation liquor used for dyeing, which was produced from sukumo (composted Polygonum indigo leaves) obtained from a craft centre in Data City, Hokkaido, Japan, by using medium containing cellulase-treated sukumo. The 16S rRNA gene sequence phylogeny suggested that Bps-1T has a distinctive position among the alkaliphilic species of the genus Bacillus, with its closest neighbours being Bacillus pseudofirmus DSM 8715T, Bacillus lindianensis DSM 26864T and Bacillus alcalophilus DSM 485T (96.1, 95.8 and 95.5 % 16S rRNA gene sequence similarities, respectively). The 16S rRNA sequence of strain Bps-1T was identical to those of strains Bps-2 and Bps-3. Cells of the novel isolate were Gram-stain-positive and were facultatively anaerobic straight rods that were motile by means of a pair of flagella (subpolar and centre sides). Spherical endospores were formed in the terminal position. Strain Bps-1T grew between 18 and 40 °C with optimum growth at 33 °C. The isolate grew in the pH range 8‒11, with optimum growth at pH 9‒10. The isoprenoid quinone detected was menaquinone-7 (MK-7), and the DNA G+C content was 40.3 %. The whole-cell fatty acid profile (>10 %) mainly consisted of anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, the isolates represent a novel species of a novel genus, for which the name Paralkalibacillus indicireducens gen. nov., sp. nov. is proposed. The type strain of this species is Bps-1T (JCM 31808T=NCIMB 15080T), with strains Bps-2 and Bps-3 representing additional strains of the species.

Bioprospecting bacterial strains for biological control of white mold on soybean

 The search for efficient biocontrol agents in agriculture has been achieved in several pathosystems. Thus, we carried out experiments under controlled conditions in greenhouse and laboratory, involving the use of bacteria Bacillus alcalophilus, Bacillus cereus GC subgrupo B, Stenotrophomonas maltophilia, Yersinia bercovieri and Photorhabdus luminescens-luminescens obtained from biofertilizers and bovine and suine manure. The bacterial strains were multiplied in nutrient-agar medium (NA) for 48 hours to evaluate the efficiency in inhibiting the mycelial growth and the development of Sclerotinia sclerotiorum in antagonism assays in Petri dishes containing BDA medium; or in aqueous suspension adjusted in 109 ufc.mL-1 by Mac Farland scale, to seeds microbiolization and spraying the aerial parts of soybean plants, totalizing three assays. The bacterial strains BB-4 (Bacillus cereus GC subgrupo B), BS-3 (Photorhabdus luminescens-luminescens), BB-1 (Bacillus alcalophilus) and BB-6 (Yersinia bercovieri) tested were shown to be effective for inhibition of mycelial growth of the pathogen, with values between 31% and 46%, and disease development in soybean seeds and plants, with values above 50% and the same levels of control treatments with thiophanate methyl + fluazinan and thiophanate methyl. These results demonstrate the presence of promising strains in control of white mold alternatively the use of chemical fungicides

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

Voltage-gated sodium channels (NaVs) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial NaVs have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial NaVs. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic NaVs NsVBa (nonselective voltage-gated Bacillus alcalophilus) and NaChBac (bacterial sodium channel from Bacillus halodurans) (IC50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of NsVBa, whereas the local anesthetic drug lidocaine was shown to antagonize NsVBa without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of NsVBa, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of NsVBa in one of the deactivated states. In mammalian NaVs, JZTx-27 preferably inhibited the inactivation of NaV1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic NaVs. More important, we proposed that JZTx-27 stabilized the NsVBa VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of NaVs.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

Ion Selectivity of the Flagellar Motors Derived from the Alkaliphilic Bacillus and Paenibacillus Species.

Many bacteria can swim using their flagella, which are filamentous organelles that extend from the cell surface. The flagellar motor is energized by either a proton (H+) or sodium ion (Na+) as the motive force. MotAB-type stators use protons, whereas MotPS- and PomAB-type stators use Na+ as the coupling ions. Recently, alkaliphilic Bacillus alcalophilus was shown to use potassium ions (K+) and rubidium ions (Rb+) for flagellar rotation, and the flagellar motor from Paenibacillus sp. TCA-20 uses divalent cations such as magnesium ions (Mg2+), calcium ions (Ca2+), and strontium ions (Sr2+) for coupling. In this chapter, we focus on how to identify the coupling ions for flagellar rotation of alkaliphilic Bacillus and Paenibacillus species.

Nonconventional cation-coupled flagellar motors derived from the alkaliphilic Bacillus and Paenibacillus species.

Prior to 2008, all previously studied conventional bacterial flagellar motors appeared to utilize either H+ or Na+ as coupling ions. Membrane-embedded stator complexes support conversion of energy using transmembrane electrochemical ion gradients. The main H+-coupled stators, known as MotAB, differ from Na+-coupled stators, PomAB of marine bacteria, and MotPS of alkaliphilic Bacillus. However, in 2008, a MotAB-type flagellar motor of alkaliphilic Bacillus clausii KSM-K16 was revealed as an exception with the first dual-function motor. This bacterium was identified as the first bacterium with a single stator-rotor that can utilize both H+ and Na+ for ion-coupling at different pH ranges. Subsequently, another exception, a MotPS-type flagellar motor of alkaliphilic Bacillus alcalophilus AV1934, was reported to utilize Na+ plus K+ and Rb+ as coupling ions for flagellar rotation. In addition, the alkaline-tolerant bacterium Paenibacillus sp. TCA20, which can utilize divalent cations such as Ca2+, Mg2+, and Sr2+, was recently isolated from a hot spring in Japan, which contains a high Ca2+ concentration. These findings show that bacterial flagellar motors isolated from unique environments utilize unexpected coupling ions. This suggests that bacteria that grow in different extreme environments adapt to local conditions and evolve their motility machinery.

Polygonibacillus indicireducens gen. nov., sp. nov., an indigo-reducing and obligate alkaliphile isolated from indigo fermentation liquor for dyeing

Obligately alkaliphilic and halophilic strains, designated In2-9T and D2-7, were isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample obtained from a craft centre in Date City, Hokkaido, Japan. The 16S rRNA gene sequence phylogeny suggested that strain In2-9T is a member of the genus Bacillus with the closest relatives being the alkaliphilic species of the genus Bacillus, Bacillus hemicellulosilyticusJCM 9152T (96.4 % 16S rRNA gene sequence similarity) and Bacillus alcalophilus DSM 485T (96.5 %). Cells of the isolate stained Gram-positive and were facultatively anaerobic straight rods that were motile by peritrichous flagella. Strain In2-9T grew between 13 and 45 °C with optimum growth at approximately 35-37 °C. The isolates grew in the pH range of 8-12 with optimum growth at pH 10. The isoprenoid quinone detected was menaquinone-6 (MK-6) and the DNA G+C content was 39.4 mol%. The whole-cell fatty acid profile mainly (>10 %) consisted of iso-C15 : 0, anteiso-C15 : 0 and C16 : 0. Spore shape and location and chemotaxonomic characteristics revealed that the isolates were distinctly different from phylogenetic neighbouring alkaliphilic species of the genus Bacillus. On the basis of phenotypic and chemotaxonomic characteristics and phylogenetic data, the isolates represent a novel species of a new genus, for which the name Polygonibacillusindicireducens gen. nov., sp. nov. is proposed. The type strain of the type species is In2-9T (=JCM 30831T=NCIMB 14982T), and strain D2-7 is an additional strain of the species.

Draft Genome Sequence of Bacillus alcalophilus AV1934, a Classic Alkaliphile Isolated from Human Feces in 1934.

Bacillus alcalophilus AV1934, isolated from human feces, was described in 1934 before microbiome studies and recent indications of novel potassium ion coupling to motility in this extremophile. Here, we report draft sequences that will facilitate an examination of whether that coupling is part of a larger cycle of potassium ion-coupled transporters.

Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus.

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; <underline>N</underline>on-<underline>s</underline>elective <underline>v</underline>oltage-gated, <underline>B</underline>acillus <underline>a</underline>lcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

Improvement of cold adaptation of Bacillus alcalophilus alkaline protease by directed evolution

Abstract Protein engineering of the Bacillus alcalophilus PB92 ATCC 31408 alkaline protease (SBA) was performed to obtain enzymes with improved cold adaptation. The activity of SBA at low temperature was enhanced through direct evolution using error-prone polymerase chain reaction. Two mutation sites, Glu110Ala and Glu134Ala, were obtained in SBA. To identify the mutation of amino acids in E110A/E134A related to its activity at low temperature, single mutants E110A and E134A were obtained via site-directed mutagenesis. The k cat / K m values of the mutants E110A, E134A and E110A/E134A at 10 °C were 1.5-, 2.2- and 2.7-fold higher, respectively, than that of the wild-type. Through the three-dimensional structure analysis, it was indicated that E110A/E134A showed an improved activity at low-temperature condition as a result of the disrupted hydrogen bond, increased protein hydrophobicity, and decreased calcium affinity. The findings of this study provides the theoretical basis and background data for improvement of the cold adaptation in SBA by protein engineering.

Wealth from waste: Optimized alkaline protease production from agro-industrial residues by Bacillus alcalophilus LW8 and its biotechnological applications

An efficient alkaline protease producer was isolated from the water of the hyperalkaline-saline Lonar soda lake and identified as Bacillus alcalophilus LW8 by culture-dependent techniques by its morphological, microscopic, biochemical, physiological and molecular characteristics. The 16S rRNA gene sequence was submitted to the GenBank nucleotide repository under accession number KC689353. Alkaline protease production was optimized by adopting a one-variable-at-a-time approach in a submerged fermentation system in modified fermentation medium (MFM). The optimized components of MFM were (w/w) casein (1%), sugarcane molasses (1%), NaCl (1%), ammonium sulphate (0.5%), KH2PO4 (0.05%), K2HPO4 (0.05%) and Na2CO3 (1%). Optimized culture conditions were used for alkaline protease production. The final yield of partially purified alkaline protease after dialysis was 53.35%. The molecular mass of the dialysed alkaline protease was 27kDa. On a Lineweaver–Burk plot, the calculated Km and Vmax values were 24mg/mL and 1000U/mg, respectively. The enzyme was remarkably stable in the pH range 7.0–12.0, with optimum activity at pH 10.0. LW8 alkaline protease was completely inhibited by phenylmethylsulphonyl fluoride at 10mmol/L, indicating that the enzyme belongs to the serine protease class. The metal ions Ca2+, Ba2+, Mg2+, Zn2+, Fe3+, Cu2+ and Mn2+ increased the catalytic activity of partially purified alkaline protease. The protease effectively decomposed the gelatinous coating on an X-ray film, hydrolysed blood clot, a blood-stain from a piece of cotton fabric and hairs from a piece of goat skin.

Sodium Dodecyl Sulphate, a Strong Inducer of Thermostable Glucanhydrolase Secretion from a Derepressed Mutant Strain of Bacillus alcalophilus GCBNA-4

In the present study, we report the optimisation of batch conditions for improved α-1,4-glucan-glucanohydrolase (GGH) secretion by a nitrous acid (NA)-treated Bacillus alcalophilus. The wild (isolate GCB-18) and NA-derivative (mutant GCBNA-4) were grown in a medium containing 10 g/L nutrient broth, 10 g/L starch, 5 g/L lactose, 2 g/L ammonium sulphate, 2 g/L CaCl2 and phosphate buffer (pH 7.6). Sodium dodecyl sulphate (SDS) was used as an enzyme inducer while batch fermentations were carried out at 40 °C. The mutant produced GGH in 40 h which was 15-fold higher than the wild in presence of SDS. Thermodynamic studies revealed that the mutant culture exhibited the capability for improved enzyme activity over a broad range of temperature (35-70 °C). The enzyme was purified by cation-exchange column chromatography with ~80 % recovery. The performance of fuzzy-logic system control was found to be highly promising for the improved substrate conversion rate. The correlation (1.045E + 0025) among variables demonstrated the model terms as highly significant indicating commercial utility of the culture used (P < 0.05).