Geobacillus Thermocatenulatus Research Articles

Pretreatment of shrimp shells with an acidic deep eutectic solvent system for chitin extraction and its enhanced performance as a carrier for immobilized lipase

Existing methods for chitin extraction usually produce substantial waste, which poses ecological hazards. Natural deep eutectic solvent (NADES) offers a promising one-step pretreatment alternative, replacing the resource-intensive demineralization (DM) and deproteinization (DP) process. Hence, in this study, the influence of various acidic NADES, on achieving a simplified one-step DM and DP in the chitin extraction process was investigated. The study yielded chitin with 87.73 % purity, and microstructural analysis showed that NADES pretreatment minimally affected chitin quality without deacetylation. In addition, chitin extracted using choline chloride-oxalic acid as a carrier displayed excellent performance in the immobilization of Geobacillus thermocatenulatus lipase 2 (GTL2) because of obvious Ca2+ activation effect. This process contributed to enhancement of immobilized enzyme activity. The immobilized GTL2 showed excellent hydrolytic capabilities, with its highest activity reaching 547.80 ± 20.62 U/mg, significantly better than the five commercial lipases that exhibited <40 % of the enzyme activity. Furthermore, the hydrolytic capacity of immobilized GTL2 was notably high for 4-nitrophenyl butyrate, measuring 935.47 ± 51.60 U/mg. This study provided a constructive approach for the one-step pretreatment of shrimp shells with organic acid-based NADES to isolate and purify chitin and its potential application as an immobilized carrier to enhance enzyme activity.

Design of a gold nanoparticles site in an engineered lipase: an artificial metalloenzyme with enantioselective reductase-like activity.

The conjugation of gold complexes with proteins has proved to be interesting and effective in obtaining artificial metalloenzymes as catalysts with improved properties such as higher stability, activity and selectivity. However, the design and precise regulation of their structure as protein nanostructured forms level remains a challenge. Here, we have designed and constructed a gold nanoparticles-enzyme bioconjugate, by tailoring the in situ formation of gold nanoparticles (AuNPs) at two specific sites on the structure of an alkalophilic lipase from Geobacillus thermocatenulatus (GTL). For this purpose, two genetically modified variants of GTL were created by inserting a unique cysteine residue into the catalytic active site by replacing the active serine (GTL-114) and into the lid site (GTL-193). The enzyme, after a first protein-gold coordination, induced the in situ formation of AuNPs, generating a homogeneous artificial enzyme. The size and morphology of the nanoparticles in the AuNPs-enzyme conjugate have been controlled by specific pH conditions in synthesis and the specific protein region where they are formed. Reductase activity of all of them was confirmed in the hydrogenation of nitroarenes in aqueous media. The protein area seemed to be key for the AuNPs, with the best TOF values obtained for the bioconjugates with AuNPs in the lid site. Finally, the protein environment and the asymmetric properties of the AuNPs were tested in the reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. A high reductive conversion and an enantiomeric excess of up to 39% towards (R)-1-phenylethanol was found using Au-Mt@GTL-114 pH 10 as a catalyst. Moderate enantioselectivity towards the opposite isomer was also observed using the Au-Mt@GTL-193 pH 10 conjugate.

Similarities of Geobacillus bacteria based on their profiles of antimicrobial susceptibility in milk samples

The genus Geobacillus is composed of thermophilic bacteria that exhibit diverse biotechnological potentialities. Specifically, Geobacillus stearothermophilus is included as a test bacterium in commercial microbiological inhibition methods, although it exhibits limited sensitivity to aminoglycosides, macrolides, and quinolones. Therefore, this article evaluates the antibiotic susceptibility profiles of five test bacteria (G. stearothermophilus subsp. calidolactis C953, Geobacillus thermocatenulatus LMG 19007, Geobacillus thermoleovorans LMG 9823, Geobacillus kaustophilus DSM 7263 and Geobacillus vulcani 13174). For that purpose, the minimum inhibitory concentrations (MICs) of 21 antibiotics were determined in milk samples for five test bacteria using the radial diffusion microbiological inhibition method. Subsequently, the similarities between bacteria and antibiotics were analyzed using cluster analysis. The dendrogram of this multivariate analysis shows an association between a group formed by G. thermocatenulatus and G. stearothermophilus and another by G. thermoleovorans, G. kaustophilus and G. vulcani. Finally, future microbiological methods could be developed in microtiter plates using G. thermocatenulatus as test bacterium, as it exhibits similar sensitivities to G. stearothermophilus. Conversely, G. vulcani, G. thermoleovorans and G. kaustophilus show higher MICs than G. thermocatenulatus.

High-Level Production of Recombinant Lipase by Fed-Batch Fermentation in Escherichia coli and Its Application in Biodiesel Synthesis from Waste Cooking Oils.

The enzymatic production of biodiesel from waste cooking oils (WCOs) offers a green and sustainable solution for the liquid fuel manufacture as well as waste resource recovery. In present study, liquid lipase was used to simplify the catalysis process, thereby reducing biodiesel production costs. An engineered Escherichia coli expressing Geobacillus thermocatenulatus lipase 2 (GTL2) was screened at an enzyme activity of 6.96 U/mg, after evaluating the propagating stability of the recombinant plasmids exceeding 86.11%. Through the beneficial feeding strategy and effective pH control, high-level production of GTL2 by fed-batch fermentation was achieved with an enzyme activity of 434.32 U/mg, which was almost 62 times that of shake flask fermentation. In addition, liquid GTL2 was used to prepare fatty acid methyl esters (FAMEs) using WCOs. The effects of the reaction time, catalyst loading, temperature, and methanol-to-oil molar ratio on FAMEs production using WCOs were explored, and a maximum FAMEs yield of 96.62% was achieved under optimized conditions. These results indicate that liquid GTL2 is a promising biocatalyst for efficient utilization of WCOs in the synthesis of biodiesel and provide a novel enzymatic process for biodiesel reducing the cost of production.

Rational engineering of low temperature activity in thermoalkalophilic Geobacillus thermocatenulatus lipase

While thermophilic enzymes have thermostability desired for broad industrial applications, they can lose activity at ambient temperatures far from their optimal temperature. Engineering cold activity into thermophilic enzymes has the potential to broaden the range of temperatures resulting in significant activity (i.e., decreasing the temperature dependence of kcat). Even though it has been widely suggested that cold activity, which results from active site flexibility, is at odds with thermostability, which results from enzyme rigidity, directed evolution experiments have shown that these properties are not mutually exclusive. In this study, rational protein engineering was used to introduce glycine as flexibility inducing mutations around the active site of Geobacillus thermocatenulatus lipase (GTL). Two mutants were found to have enhanced specific activity compared to wild-type at temperatures between 283 K and 363 K with p-nitrophenol butyrate but not with larger substrates. Kinetics assay revealed both mutations resulted in psychrophilic traits, such as lower activation enthalpy and more negative entropy values compared to wild type in all substrates. Furthermore, the mutants had significantly improved thermostability compared to wild type enzyme, which proves that it is feasible to improve the cold activity without trade-off. Our study provides insight into the enzyme cold adaptation mechanism and design principles for engineering cold activity into thermostable enzymes.

Identification and Characterization of a Novel Thermostable GDSL-Type Lipase from Geobacillus thermocatenulatus.

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDSPAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50°C and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and in silico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

High-efficiency expression of the thermophilic lipase from Geobacillus thermocatenulatus in Escherichia coli and its application in the enzymatic hydrolysis of rapeseed oil.

Long-chain fatty acids are widely used in food and chemical industries, and the enzymatic preparation of fatty acids is considered an environmentally friendly process. In the present study, long-chain fatty acids were prepared by the enzymatic hydrolysis of rapeseed oil with a genetically engineered lipase. Because thermophilic lipase has strong stability at higher temperatures, it was more suitable for the industrial production of long-chain fatty acids. Therefore, the thermophilic lipase BTL2 from Geobacillus thermocatenulatus was efficiently expressed in E. coli BL21(DE3) cells with an enzyme activity of 39.50 U/mg followed by gene codon optimisation. Experimental results showed that the recombinant lipase BTL2 exhibited excellent resistance to certain organic solvents (n-hexane, benzene, ethanol, and butanol). The metal cation Ca2+ and the non-ionic surfactant Triton-100X enhanced enzyme activity by 7.36% and 56.21% respectively. Moreover, the acid value of the liberated long-chain fatty acids by hydrolysing rapeseed oil was approximately 161.64 mg KOH/g at 50 °C in 24 h, the hydrolytic conversion rate was 91.45%, and the productivity was approximately 6.735 mg KOH/gh. These results suggested that the recombinant lipase BTL2 has excellent hydrolytic performance for rapeseed oil and showed great potential for the enzymatic preparation of long-chain fatty acids.

Microbial Biodegradation of Paraffin Wax in Malaysian Crude Oil Mediated by Degradative Enzymes.

The deposition of paraffin wax in crude oil is a problem faced by the oil and gas industry during extraction, transportation, and refining of crude oil. Most of the commercialized chemical additives to prevent wax are expensive and toxic. As an environmentally friendly alternative, this study aims to find a novel thermophilic bacterial strain capable of degrading paraffin wax in crude oil to control wax deposition. To achieve this, the biodegradation of crude oil paraffin wax by 11 bacteria isolated from seawater and oil-contaminated soil samples was investigated at 70°C. The bacteria were identified as Geobacillus kaustophilus N3A7, NFA23, DFY1, Geobacillus jurassicus MK7, Geobacillus thermocatenulatus T7, Parageobacillus caldoxylosilyticus DFY3 and AZ72, Anoxybacillus geothermalis D9, Geobacillus stearothermophilus SA36, AD11, and AD24. The GCMS analysis showed that strains N3A7, MK7, DFY1, AD11, and AD24 achieved more than 70% biodegradation efficiency of crude oil in a short period (3 days). Notably, most of the strains could completely degrade C37–C40 and increase the ratio of C14–C18, especially during the initial 2 days incubation. In addition, the degradation of crude oil also resulted in changes in the pH of the medium. The degradation of crude oil is associated with the production of degradative enzymes such as alkane monooxygenase, alcohol dehydrogenase, lipase, and esterase. Among the 11 strains, the highest activities of alkane monooxygenase were recorded in strain AD24. A comparatively higher overall alcohol dehydrogenase, lipase, and esterase activities were observed in strains N3A7, MK7, DFY1, AD11, and AD24. Thus, there is a potential to use these strains in oil reservoirs, crude oil processing, and recovery to control wax deposition. Their ability to withstand high temperature and produce degradative enzymes for long-chain hydrocarbon degradation led to an increase in the short-chain hydrocarbon ratio, and subsequently, improving the quality of the oil.

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

Abstract. 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.

Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2.

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg−1) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg−1), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.

N-Terminal Domain Truncation and Domain Insertion-Based Engineering of a Novel Thermostable Type I Pullulanase from Geobacillus thermocatenulatus.

A novel thermostable type I pullulanase gene ( pul GT) from Geobacillus thermocatenulatus DSMZ730 was cloned. It has an open reading frame of 2154 bp encoding 718 amino acids. G. thermocatenulatus pullulanase (PulGT) was found to be optimally active at pH 6.5 and 70 °C. It exhibited stable activity in the pH range of 5.5-7.0. PulGT lacked three domains (CBM41 domain, X25 domain, and X45 domain) compared with the pullulanase from Bacillus acidopullulyticus ( 2WAN ). Different N-terminally domain truncated (730T) or spliced (730T-U1 and 730T-U2) mutants were constructed. Truncating the N-terminal 85 amino acids decreased the Km value and did not change its optimum pH, an advantageous biochemical property in some applications. Compared with 2WAN , PulGT can be used directly for maize starch saccharification without adjusting the pH, which reduces cost and improves efficiency.

Ultra-Small Pd(0) Nanoparticles into a Designed Semisynthetic Lipase: An Efficient and Recyclable Heterogeneous Biohybrid Catalyst for the Heck Reaction under Mild Conditions.

A novel heterogeneous enzyme-palladium (Pd) (0) nanoparticles (PdNPs) bionanohybrid has been synthesized by an efficient, green, and straightforward methodology. A designed Geobacillus thermocatenulatus lipase (GTL) variant genetically and then chemically modified by the introduction of a tailor-made cysteine-containing complementary peptide- was used as the stabilizing and reducing agent for the in situ formation of ultra-small PdNPs nanoparticles embedded on the protein structure. This bionanohybrid was an excellent catalyst in the synthesis of trans-ethyl cinnamate by Heck reaction at 65 °C. It showed the best catalytic performance in dimethylformamide (DMF) containing 10–25% of water as a solvent but was also able to catalyze the reaction in pure DMF or with a higher amount of water as co-solvent. The recyclability and stability were excellent, maintaining more than 90% of catalytic activity after five cycles of use.

Investigating the structural properties of the active conformation BTL2 of a lipase from Geobacillus thermocatenulatus in toluene using molecular dynamic simulations and engineering BTL2 via in-silico mutation.

The discovery or development of thermoalkalophilic lipases that show high levels of catalytic activity in organic solvents would have important industrial ramifications. However, this goal is yet to be achieved because organic solvents induce structural changes in lipases that suppress their catalytic abilities. A deep understanding of these structural changes to lipases in the presence of organic solvents is required before strategies can be devised to stop them from occurring. In this work, we investigated the effects of an organic reaction medium, toluene, on the structure of the Bacillus thermocatenulatus lipase BTL2 using MD simulation. The main aims were to identify the regions of the protein that are particularly sensitive to the presence of an organic solvent, and how the presence of a hydrophobic medium affects the overall stability of the enzyme. Upon analyzing how the behavior of the enzyme differed in aqueous and hydrophobic media, it was found that many significant zones of the protein suffer in the presence of an organic solvent, which increases the rigidity of the system. This was readily apparent when we investigated important noncovalent interactions (salt bridges) and probed how distances between the atoms of the catalytic triad Ser114, Asp318, and His359 change in the presence of toluene. Moreover, the high tendency for the system to destabilize in toluene was explained by the results of FoldX calculations. Calculations showed that the addition of a small amount of water to the hydrophobic reaction environment should restore the required flexibility of BTL2. The insights gained from the analysis of our simulations allowed us to propose a modification of BTL2, the G116P mutation, that should result in the structural behavior of BTL2 in organic solvent being closer to that of BTL2 in water.

Effect of Site-Specific Peptide-Tag Labeling on the Biocatalytic Properties of Thermoalkalophilic Lipase from Geobacillus thermocatenulatus.

Tailor-made peptides were investigated for site-specific tag labeling of Geobacillus thermocatenulatus lipase (GTL). GTL was first genetically modified by introducing a unique cysteine on the lid site of the enzyme to produce two variants (GTLσ-A193C and GTLσ-S196C). Chemical modification was performed by using a small library of cysteine-containing peptides. The synthesized peptide-lipase biocatalysts were highly stable, more active, more specific, and more selective toward different substrates than unmodified GTL. Very high enzyme thermostability of GTLσ-A193C modified with peptides Ac-Cys-Phe-Gly-Phe-Gly-Phe-CONH2 (1) and Ac-Cys-Phe-Phe-CONH2 (2) (>95 % activity after 24 h at 60 °C) was observed. The incorporation of 1 and 2 in GTLσ-S196C improved its catalytic activity in the hydrolysis of p-nitrophenyl butyrate by factors of three and greater than five, respectively. The specificity for short-chain versus long-chain esters was also strongly improved. The diacylglycerol activity of GTLσ-S196C was enhanced more than tenfold by the incorporation of 1 and more than threefold by modification of this variant with Ac-Cys-(Arg)7 -CONH2 (6) in the hydrolysis of 1-stearoyl-2-arachidonoyl-sn-glycerol. The enantioselectivity of GTLσ-S196C increased for all formed bioconjugates, and the GTLσ-S196C-1 conjugate was the most active and selective in the hydrolysis of dimethylphenyl glutarate at pH 7 (72 % ee), also showing an inversion in the enzyme enantiopreference.

New Strategy for the Immobilization of Lipases on Glyoxyl-Agarose Supports: Production of Robust Biocatalysts for Natural Oil Transformation.

Immobilization on Glyoxyl–agarose support (Gx) is one of the best strategies to stabilize enzymes. However, the strategy is difficult to apply at neutral pH when most enzymes are stable and, even when possible, produces labile derivatives. This work contributes to overcoming this hurdle through a strategy that combines solid-phase amination, presence of key additives, and derivative basification. To this end, aminated industrial lipases from Candida artarctica (CAL), Thermomyces lunuginosus (TLL), and the recombinant Geobacillus thermocatenulatus (BTL2) were immobilized on Gx for the first time at neutral pH using anthranilic acid (AA) or DTT as additives (immobilization yields >70%; recovered activities 37.5–76.7%). The spectroscopic evidence suggests nucleophilic catalysis and/or adsorption as the initial lipase immobilization events. Subsequent basification drastically increases the stability of BTL2–glyoxyl derivatives under harsh conditions (t1/2, from 2.1–54.5 h at 70 °C; from 10.2 h–140 h in 80% dioxane). The novel BTL2-derivatives were active and selective in fish oil hydrolysis (1.0–1.8 μmol of polyunsaturated fatty acids (PUFAs) min−1·g−1) whereas the selected TLL-derivative was as active and stable in biodiesel production (fatty ethyl esters, EE) as the commercial Novozyme®-435 after ten reaction cycles (~70% EE). Therefore, the potential of the proposed strategy in producing suitable biocatalysts for industrial processes was demonstrated.

Properties of a novel thermostable glucose isomerase mined from Thermus oshimai and its application to preparation of high fructose corn syrup.

Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95°C and could retain more than 80% of initial activity in the presence of 20mM Mn2+ at 85°C for 48h. The Km and kcat/Km values for ToGI were 81.46mM and 21.77min-1mM-1, respectively. Furthermore, the maximum conversion yield of 400g/L d-glucose to d-fructose at 85°C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature.

Semisynthetic Enzymes by Protein-Peptide Site-Directed Covalent Conjugation: Methods and Applications.

This chapter describes the rational design and synthesis of semisynthetic lipases by site-directed incorporation of tailor-made peptides on the lipase-lid site to improve its activity, specificity, and enantioselectivity in specific biotransformations. Cysteine was genetically introduced at a particular point of the oligopeptide lid of the enzyme, and cysteine-containing peptides, complementary to the amino acid sequence on the lid site of Geobacillus thermocatenulatus lipase (BTL), were covalently attached on the lid of two different cysteine-BTL variants based on a fast thiol-disulfide exchange ligation followed by desulfurization. The BTL variants were initially immobilized on solid support to introduce the advantages of solid-state chemistry, such as quantitative transformations, easy purification, and recyclability. In the two different immobilized variants BTL-A193C and BTL-L230C, the cysteine was then activated with 2-dipyridyldisulfide to help the disulfide exchange with the peptide, generating the semisynthetic enzyme in high yield. Excellent results of improvement of activity and selectivity were obtained. For example, the peptide-BTL conjugate (at position 193) was 40-fold more active than the corresponding unmodified enzyme for the hydrolysis of per-acetylated thymidine at pH 5, or fourfold in the desymmetrization of dimethyl-3-phenylglutarate at pH 7. The new enzyme also exhibited excellent enantioselectivity in the desymmetrization reaction with enantiomeric excess (ee) of >99% when compared to that of the unmodified enzyme (ee=78%).

Combinatorial library strategy for strong overexpression of the lipase from Geobacillus thermocatenulatus on the cell surface of yeast Pichia pastoris

The yeast cell surface display technique allows for the expression of a target protein on the yeast cell surface and has many applications such as the immobilization of enzymes and the development of biosensors. To increase the expression of the BTL2, a lipase from Geobacillus thermocatenulatus, on the cell surface of yeast Pichia pastoris, we developed a combinatorial library strategy for selecting appropriate expression cassette comprising sequences encoding a promoter, secretion signal, mature BTL2, anchoring protein, and terminator. The transformant GS115/D90, which comprised P. pastoris ENO1 promoter sequence, Hansenula polymorpha GAS1 secretion signal sequence, and Saccharomyces cerevisiae GAS1 anchoring protein gene, exhibited 5-fold higher lipase activity compared to the control strain harboring a conventional expression cassette. Using the developed strategy, an appropriate expression cassette for the strong overexpression of target proteins on the cell surface of yeast could be rapidly and easily obtained.

Asymmetric hydrolysis of dimethyl-3-phenylglutarate in sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase

The main goal of this work was to study the stereoselective behavior of immobilized Geobacillus thermocatenulatus lipase (BTL2) in a sequential batch reactor using the partial and asymmetric hydrolysis of dimethyl-3 phenylglutarate (DMFG) as a model reaction. To reach this goal, BTL2 lipase was immobilized on Sepharose and silica supports with cyanogen bromide and octyl groups (monofunctional supports) and undecanol-glyoxyl and octyl-epoxides groups (heterofunctional supports), to determine the effect of the enzyme orientation during the immobilization process on their catalytic properties. In the hydrolysis of DMFG, the biocatalyst obtained with undecanol-glyoxyl Sepharose proved to be the most stereolective with an enantiomeric excess (e.e.) value of 90% in aqueous media. This behavior can be attributed to differences in the orientation of the lipase on the support. In sequential batch reactor operation, the e.e. remained constant in the first two batches; however, from the third batch on the e.e. decreased slightly maybe due to a change in the conformation of the enzyme at the reaction conditions. Finally, the high purity S-methyl-3-phenyl glutarate produced in sequential batch reactor operation shows that the biocatalyst can be reused at least twice without losing stereoselectivity, favoring a reduction in the process cost.

Isolation and characterization of thermophilic bacterial strains from Soldhar (Tapovan) hot spring in Central Himalayan Region, India

In the present investigation, 11 morphologically distinct thermophilic bacterial strains have been isolated from the Soldhar hot spring site in the Garhwal region of the Indian Himalaya. The phenotypic and genotypic characters of the isolates were studied using standards methods. All of them developed circular colonies on Tryptone Soy agar plates at 70 °C following 24 h incubation. The isolates grew over a wide range of temperatures (20–100 °C) and pHs (5–10). The cell morphology varied from short to long rods and cocci with a dominance of Gram-positive bacteria. The isolates exhibited varied biochemical properties with regard to the production of various enzymes and capability to ferment sugars. Based on restriction digestion with HaeIII, six phylogenetic groups were formed. Two isolates were selected for 16S rRNA gene sequence analysis, and exhibited maximum similarity and closest homology with Geobacillus thermocatenulatus and Geobacillus thermoleovorans, respectively.