Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from ...psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast,
Glaciozyma antarctica
. A cDNA encoding the antifreeze protein,
AFP4
, from
G. antarctica
PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from
Leucosporidium
sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in
Escherichia coli
resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.
New thermostable β-1,3–1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis ...predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 μmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.
•Alkaliphilic Bacillus lehensis G1 produced alkaline β-1,3–1,4- glucanase.•Overexpression of β-1,3–1,4- glucanase using pET200 and E. coli system.•Blg29 was produced as a soluble protein.•Blg29 is classified as thermostable Lichenase.
Hazardous micropollutants (MPs) such as pharmaceutically active compounds (PhACs), pesticides and personal care products (PCPs) have emerged as a critical concern nowadays for acquiring clean and ...safe water resources. In the last few decades, innumerable water treatment methods involving biodegradation, adsorption and advanced oxidation process have been utilized for the removal of MPs. Of these methods, membrane technology has proven to be a promising technique for the removal of MPs due to its sustainability, high efficiency and cost-effectiveness. Herein, the aim of this article is to provide a comprehensive review regarding the MPs rejection mechanisms of reverse osmosis (RO) and nanofiltration (NF) membranes after incorporation of nanomaterials and also surface modification atop the PA layer. Size exclusion, adsorption and electrostatic charge interaction mechanisms play important roles in governing the MP removal rate. In addition, this review also discusses the state-of-the-art research on the surface modification of thin film composite (TFC) membrane and nanomaterials-incorporated thin film nanocomposite (TFN) membrane in enhancing MPs removal performance. It is hoped that this review can provide insights in modifying the physicochemical properties of NF and RO membranes to achieve better performance in water treatment process, particularly for the removal of emerging hazardous substances.
Schematic illustration of surface-modified TFC membrane and nanomaterials-incorporated TFN membrane against MPs compounds. Display omitted
•Occurrence, risks and type of emerging micropollutants (MPs) have been identified.•Removal of emerging MPs via RO and NF membranes have been summarized.•TFN and surface-modified TFC membranes showed remarkable MPs removal rate.•The MPs rejection mechanisms of polyamide RO/NF membranes were elucidated.
Psychrophilic organisms possess several adaptive strategies which allow them to sustain life at low temperatures between −20 to 20 °C. Studies on Antarctic psychrophiles are interesting due to the ...multiple stressors that exist on the permanently cold continent. These organisms produce, among other peculiarities, cold-active enzymes which not only have tremendous biotechnological potential but are valuable models for fundamental research into protein structure and function. Recent innovations in omics technologies such as genomics, transcriptomics, proteomics and metabolomics have contributed a remarkable perspective of the molecular basis underpinning the mechanisms of cold adaptation. This review critically discusses similar and different strategies of cold adaptation in the obligate psychrophilic yeast, Glaciozyma antarctica PI12 at the molecular (genome structure, proteins and enzymes, gene expression) and physiological (antifreeze proteins, membrane fluidity, stress-related proteins) levels. Our extensive studies on G. antarctica have revealed significant insights towards the innate capacity of- and the adaptation strategies employed by this psychrophilic yeast for life in the persistent cold. Furthermore, several cold-active enzymes and proteins with biotechnological potential are also discussed.
Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to ...enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.
Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast,
...Glaciozyma antarctica
predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from
Coniophora puteana
. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an
Escherichia coli
expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50% residual activity). Its
K
m
value was 256 μM and its catalytic efficiency was 81.7 s
−1
. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.
The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization ...inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.
The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action ...of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults.
Antifreeze proteins (AFPs) are unique proteins that have an affinity toward ice. Due to their potential applications in the food and medical industries, many attempts have been made to produce AFPs ...in large quantities. In this study, recombinant Afp1 from
Glaciozyma antarctica
, a psychrophilic yeast, was overexpressed in a methylotrophic yeast,
Pichia pastoris
, followed by 5-L fermenter expression. The highest yield of Afp obtained from the fermentation was 39.5 mg/L when the cells were cultivated at
16
∘
C
and at pH 5.0. Afp1 was produced as a glycoprotein (
∼
55 kDa) based on gel staining using a glycoprotein kit. Antifreeze activities of the recombinant Afp1 were exhibited through thermal hysteresis (TH) and recrystallization inhibition (RI) where the highest TH value recorded was at
∼
0.5
∘
C
at 10 mg/mL. This value is higher when compared to the recombinant Afp1 produced in
Escherichia coli
(
0.08
∘
C
) as well as the native antifreeze protein from
G. antarctica
(
0.1
∘
C
). Both TH and RI activities increased when higher protein concentrations were used. Effects of temperature on stability showed that Afp1 had lost its activity after being incubated at a temperature higher than
20
∘
C
. The cryoprotective effects of Afp1 on cellulases showed that the treated cellulase retained up to
∼
20% of its activity following several cycles of freeze–thawing. This indicates that Afp1 might act as a cryoprotective agent and has the potential for use in biotechnology applications.
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