Draft Genome Analysis of Acinetobacter indicus Strain UBT1, an Efficient Lipase and Biosurfactant Producer.

Acinetobacter indicus strain UBT1 has shown efficient lipase (243 U ml-1) and biosurfactant (61.1% E24% emulsification and surface tension reduction to 37.7 mN m-1) production capabilities using agro-industrial waste as sole carbon source. We report here the draft genome sequence of A. indicus strain UBT1 having genome size of 2.97 Mb with 45.90% GC content. Total 2721 coding genes were predicted using National Center for Biotechnology Information-Prokaryotic Genome Annotation Pipeline (NCBI-PGAP). The whole genome shotgun project sequence data are accessible through NCBI Gene Bank under accession no. JABFOI000000000. PGAP annotation revealed the presence of the triacylglycerol lipase, phospholipase etc., that circuitously confers the oil consumption competency to the strain UBT1. Rapid Annotation using the Subsystem Technology (RAST) server used for mapping the genes to the subsystem resulted in 278 subsystem with 30% subsystem coverage. The draft genome data can be used to exploit the A. indicus strain UBT1 for its advance biotechnological application and also for further comparative genomic studies.

Draft genome sequence of a thermostable, alkaliphilic α-amylase and protease producing Bacillus amyloliquefaciens strain KCP2.

Bacillus amyloliquefaciens strain KCP2 was isolated from municipal food waste samples collected in Vallabh Vidyanagar, Gujarat, India. Strain KCP2 is noteworthy due to its ability to produce a thermostable, alkaliphilic α-amylase and a protease. These enzymes have importance in several industrial processes including bread making, brewing, starch processing, pharmacy, and textile industries. Whole genome sequencing of strain KCP2 showed that the estimated genome size was 3.9 Mb, the G + C content was 46%, and it coded for 4113 genes.

ß-Cyclodextrin Production by Cyclodextrin Glucanotransferase from an Alkaliphile Microbacterium terrae KNR 9 Using Different Starch Substrates.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an important member of α-amylase family which can degrade the starch and produce cyclodextrins (CDs) as a result of intramolecular transglycosylation (cyclization). ß-Cyclodextrin production was carried out using the purified CGTase enzyme from an alkaliphile Microbacterium terrae KNR 9 with different starches in raw as well as gelatinized form. Cyclodextrin production was confirmed using thin layer chromatography. Six different starch substrates, namely, soluble starch, potato starch, sago starch, corn starch, corn flour, and rice flour, were tested for CD production. Raw potato starch granules were found to be the best substrate giving 13.46 gm/L of cyclodextrins after 1 h of incubation at 60°C. Raw sago starch gave 12.96 gm/L of cyclodextrins as the second best substrate. To achieve the maximum cyclodextrin production, statistical optimization using Central Composite Design (CCD) was carried out with three parameters, namely, potato starch concentration, CGTase enzyme concentration, and incubation temperature. Cyclodextrin production of 28.22 (gm/L) was achieved with the optimized parameters suggested by the model which are CGTase 4.8 U/L, starch 150 gm/L, and temperature 55.6°C. The suggested optimized conditions showed about 15% increase in ß-cyclodextrin production (28.22 gm/L) at 55.6°C as compared to 24.48 gm/L at 60°C. The degradation of raw potato starch granules by purified CGTase was also confirmed by microscopic observations.

Screening and Selection of Medium Components for Cyclodextrin Glucanotransferase Production by New Alkaliphile Microbacterium terrae KNR 9 Using Plackett-Burman Design.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) production using new alkaliphile Microbacterium terrae KNR 9 was investigated by submerged fermentation. Statistical screening for components belonging to different categories, namely, soluble and raw starches as carbon sources, complex organic and inorganic nitrogen sources, minerals, a buffering agent, and a surfactant, has been carried out for CGTase production using Plackett-Burman factorial design. To screen out k (19), number of variables, k + 1 (20), number of experiments, were performed. Among the fourteen components screened, four components, namely, soluble starch, corn flour, yeast extract, and K2HPO4, were identified as significant with reference to their concentration effect and corresponding p value. Although soluble starch showed highest significance, comparable significance was also observed with corn flour and hence it was selected as a sole carbon source along with yeast extract and K2HPO4 for further media optimization studies. Using screened components, CGTase production was increased to 45% and 87% at shake flask level and laboratory scale fermenter, respectively, as compared to basal media.

A novel cyclodextrin glucanotransferase from an alkaliphile Microbacterium terrae KNR 9: purification and properties.

Cyclodextrin glucanotransferase (CGTase, EC. 2.1.1.19) produced using new alkaliphile Microbacterium terrae KNR 9 has been purified to homogeneity in a single step by the starch adsorption method. The specific activity of the purified CGTase was 45 U/mg compared to crude 0.9 U/mg. This resulted in a 50-fold purification of the enzyme with 33 % yield. The molecular weight of the purified enzyme was found to be 27.72 kDa as determined by SDS-PAGE. Non-denaturing gel electrophoresis and activity staining confirmed the presence of CGTase in crude and the ammonium sulfate precipitate fraction. The purified CGTase has a pI value of 4.2. The optimum pH of 6.0 and 60 °C temperature were found to be the best for CGTase activity. Purified CGTase showed 5.18 kcal/mol activation energy (Ea). The CGTase activity was increased in the presence of metal ions (5 mM): Ca+2 (130 %), Mg+2 (123 %), Mn+2 (119 %) and Co+2 (116 %). The enzyme activity was strongly inhibited in the presence of Hg+2 (0.0 %), Cu+2 (0.0 %) and Fe+2 (3.8 %). Inhibitor N-bromosuccinimide (5 mM) showed the highest 96 % inhibition of CGTase activity. SDS and triton X-100 among different detergents and surfactants (1.0 %, w/v) tested showed 92 % inhibition. Among the organic solvents checked for their effect on enzyme activity, 5 % (v/v) toluene resulted in 48 % increased activity. Polyethylene glycol-6000 showed a 26 % increase in the CGTase activity. The kinetic parameters K m and V max were 10 mg/ml and 146 µmol/mg min, respectively, for purified CGTase.

A statistical approach for the production of thermostable and alklophilic alpha-amylase from Bacillus amyloliquefaciens KCP2 under solid-state fermentation.

The bacterial strain producing thermostable, alklophilic alpha-amylase was identified as Bacillus amyloliquefaciens KCP2 using 16S rDNA gene sequencing data (NCBI Accession No: KF112071). Medium components were optimized through the statistical approach for the synthesis of alpha-amylase by the organism under solid-state fermentation using wheat bran as the substrate. The medium components influencing the enzyme production were identified using a two-level fractional factorial Plackett-Burman design. Among the various variables screened, starch, ammonium sulphate and calcium chloride were found to be most significant medium components. The optimum levels of these significant parameters were determined employing the response surface Central Composite design which significantly increased the enzyme production with the supplementation of starch 0.01 g, ammonium sulphate 0.2 g and 5 mM calcium chloride in the production medium. Temperature and pH stability of the alpha-amylase suggested its wide application in the food and pharmaceutical industries.

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1.

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol(-1) while temperature quotient (Q 10 ) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40-50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa1 and pKa2 of ionisable groups of active site controlling V max were 3.5 and 6.8, respectively. V max , K m and K cat for starch hydrolysis were found to be 58.82 U ml(-1), 1.17 mg (starch) ml(-1) and 449 s(-1), respectively. Activation energy for irreversible inactivation (E a(d)) of glucoamylase was 74.85 kJ mol(-1). Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.