Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.

Kinetic Characterization of β-xylosidase (GbtXyl43A) from Wild-type Geobacillus thermoleovorans IT-08 and The Variant GbtXyl43A-D121N

GbtXyl43A, a β-xylosidase that is isolated from Geobacillus thermoleovorans IT-08 and grouped in GH43 family. The substitution of 121Asp residue with Asn in GbtXyl43A caused decrease the enzyme activity. The aim of this study, determine the kinetic characteristics of wild-type GbtXyl43A and D121N variant using Vmax, KM, kcat, and kcat/KM. These parameters indicated catalytic mechanism of GbtXyl43A and its derivative. All of them were produced in Escherichia coli BL21 star. The purification of wild-type GbtXyl43A using affinity chromatography, but D121N variant also required anion-exchange chromatography. The specific activity of wild-type GbtXyl43A and D121N variant were 0.471 U mg-1 in purity level 55,44 and 0.012 U mg-1 in purity level 2,407, respectively. Both enzymes had same molecular weight, ~58 kDa. The kinetic parameters of wild-type GbtXyl43A were KM: 2.845 mM, kcat: 0.033 s-1, Vmax: 0.0033 mM min-1and kcat/KM: 0.0115 s-1mM-1. Furthermore, the KM, kcat, Vmax, and kcat/KM values of D121N variant were 4.565 mM, 1.01 × 10-4 mM min-1, 0.140 × 10-4 s-1, and 0.0307 s-1mM-1, respectively. The KM value of the D121N variant was higher than its wild type and showed the affinity of D121N variant was lower than GbtXyl43A

Whole-Genome Sequence of Geobacillus thermoleovorans ARTRW1, Isolated from Armutlu Geothermal Spring, Turkey

ABSTRACT The thermophilic microorganism Geobacillus thermoleovorans ARTRW1 was isolated from water samples collected in the Armutlu hot spring in Turkey. Here, the whole-genome sequence and its annotations are reported.

Short Communication: Preliminary phylogenetic analysis of bacteria producing laccase isolated from Gunung Pancar, Bogor, Indonesia

Mar WW, Rohman A, Muwafiqi NH, Laras GA, Agustina D, Asmarani O, Puspaningsih NNT. 2020. Short Communication: Preliminary phylogenetic analysis of bacteria producing laccase isolated from Gunung Pancar, Bogor, Indonesia. Biodiversitas 21: 2113-2118. Interpretation of phylogenetic trees is essential in understanding relationships between organisms, as well as their characteristics, bionetwork, and even their genomic and developmental ecology. The aim of this research is to analyze the phylogenetic bacteria producing laccase isolated from Gunung Pancar Bogor, Indonesia. Phylogenetic analysis of Geobacillus kaustophilus TP-02 producing laccase was performed using alignments with all Bacillus clusters. A phylogenetic tree was constructed by the neighbor-joining (NJ) method, using the maximum likelihood parameter. Laccase was purified using ammonium sulfate precipitation to 2.67-fold. The activity of crude laccase was determined to be 93.39 U/ml using 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Moreover, Geobacillus related to counting some Geobacillus genus such as Geobacillus thermocatenulatus, Geobacillus sp. MAS1, Geobacillus thermoleovorans, and Geobacillus zalihae, having similarity under 91% with comparison to the Geobacillus kaustophilius sequence in GenBank. Therefore, results show that G. kaustophilus TP-02 does not have any closeness with other Geobacillus genera, even though these species have the same ancestor.

Novel Geobacillus thermoleovorans KNG 112 Thermophilic Bacteria from Bandaru Hot Spring: A Potential Producer of Thermostable enzymes.

Amylase and lipase producing novel bacterium (strain IC5) were isolated from Bandaru hot spring, Karnataka, India. The cell was found to be thermophilic, gram-positive, aerobic, non-motile, capable of growing at different optimum parameters of pH 7, temperature 55°C and tolerated maximally 0-8% (w/v) NaCl, which has the ability to show good amylotic and lipolytic activities. Phylogenetic analysis of the bacterium using the 16S rRNA gene was revealed that the strain belongs to genus Geobacillus. The isolated strain IC5 was in close resemblance with the gene of Geobacillus thermoleovorans EC-5 having 99% of similarities. During the production of amylase, the maximum activity was found when temperature and pH ranged from 50 to 60 ºC and from 7 to 8 respectively. The strain used starch as a carbon source with an agitation speed of 120rpm for maximal amylase production.