Regulation of terpenoid biosynthesis by miRNA in Persicaria minor induced by Fusarium oxysporum.

BACKGROUND: Persicaria minor (kesum) is an herbaceous plant with a high level of secondary metabolite compounds, particularly terpenoids. These terpenoid compounds have well-established roles in the pharmaceutical and food industries. Although the terpenoids of P. minor have been studied thoroughly, the involvement of microRNA (miRNA) in terpenoid regulation remains poorly understood and needs to be explored. In this study, P. minor plants were inoculated with the pathogenic fungus Fusarium oxysporum for terpenoid induction. RESULT: SPME GC-MS analysis showed the highest terpenoid accumulation on the 6th day post-inoculation (dpi) compared to the other treatment time points (0 dpi, 3 dpi, and 9 dpi). Among the increased terpenoid compounds, α-cedrene, valencene and ß-bisabolene were prominent. P. minor inoculated for 6 days was selected for miRNA library construction using next generation sequencing. Differential gene expression analysis showed that 58 miRNAs belonging to 30 families had significantly altered regulation. Among these 58 differentially expressed genes (DEGs), 27 [corrected] miRNAs were upregulated, whereas 31 [corrected] miRNAs were downregulated. Two putative novel pre-miRNAs were identified and validated through reverse transcriptase PCR. Prediction of target transcripts potentially involved in the mevalonate pathway (MVA) was carried out by psRobot software, resulting in four miRNAs: pmi-miR530, pmi-miR6173, pmi-miR6300 and a novel miRNA, pmi-Nov_13. In addition, two miRNAs, miR396a and miR398f/g, were predicted to have their target transcripts in the non-mevalonate pathway (MEP). In addition, a novel miRNA, pmi-Nov_12, was identified to have a target gene involved in green leaf volatile (GLV) biosynthesis. RT-qPCR analysis showed that pmi-miR6173, pmi-miR6300 and pmi-nov_13 were downregulated, while miR396a and miR398f/g were upregulated. Pmi-miR530 showed upregulation at 9 dpi, and dynamic expression was observed for pmi-nov_12. Pmi-6300 and pmi-miR396a cleavage sites were detected through degradome sequence analysis. Furthermore, the relationship between miRNA metabolites and mRNA metabolites was validated using correlation analysis. CONCLUSION: Our findings suggest that six studied miRNAs post-transcriptionally regulate terpenoid biosynthesis in P. minor. This regulatory behaviour of miRNAs has potential as a genetic tool to regulate terpenoid biosynthesis in P. minor.

Correction to: Regulation of terpenoid biosynthesis by miRNA in Persicaria minor induced by Fusarium oxysporum.

Following publication of the original article [1], the authors reported a number of errors, which are listed in this Correction article. The corrections are marked in bold.

Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger.

Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg-1 and a low Km value of 1.88 mM when p-nitrophenyl-ß-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10-4 mM-1s-1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg-1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.

Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica.

Dienelactone hydrolase, an α/ß 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/ß hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/ß 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 Km 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.

Production of an oligosaccharide-specific cellobiohydrolase from the thermophilic fungus Thielavia terrestris.

OBJECTIVES: To express and determine the hydrolytic activity of a cellobiohydrolase (TTCBH6B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris. RESULTS: Ttcbh6B encodes a protein of 507 amino acid residues with a predicted molecular mass of 54 kDa. TTCBH6B contains a familial 6-glycosyl hydrolase catalytic domain and a type I carbohydrate-binding module. TTCBH6B was expressed and purified to homogeneity but the purified enzyme was inactive against Avicel. It could, however, digest Celluclast-treated Avicel producing cellobiose (0.27 µmol min(-1) mg(-1)). To determine the substrate preferences of TTCBH6B, oligosaccharides of varying numbers of subunits were generated by acid hydrolysis of Avicel and fluorescently tagged. Peaks corresponding to oligosaccharides containing three to six glucose units were reduced to cellobiose after addition of TTCBH6B. CONCLUSION: TTCBH6B is active against shorter oligosaccharides rather than polysaccharides.

A Sco protein among the hypothetical proteins of Bacillus lehensis G1: Its 3D macromolecular structure and association with Cytochrome C Oxidase.

BACKGROUND: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) which are largely non-similar to other known, well-characterized proteins. Predicting and solving their structures and functions is imperative to aid understanding of any given organism as a complete biological system. The present study highlights the primary effort to classify and cluster 1202 HPs of Bacillus lehensis G1 alkaliphile to serve as a platform to mine and select specific HP(s) to be studied further in greater detail. RESULTS: All HPs of B. lehensis G1 were grouped according to their predicted functions based on the presence of functional domains in their sequences. From the metal-binding group of HPs of the cluster, an HP termed Bleg1_2507 was discovered to contain a thioredoxin (Trx) domain and highly-conserved metal-binding ligands represented by Cys69, Cys73 and His159, similar to all prokaryotic and eukaryotic Sco proteins. The built 3D structure of Bleg1_2507 showed that it shared the ßαßαßß core structure of Trx-like proteins as well as three flanking ß-sheets, a 310 -helix at the N-terminus and a hairpin structure unique to Sco proteins. Docking simulations provided an interesting view of Bleg1_2507 in association with its putative cytochrome c oxidase subunit II (COXII) redox partner, Bleg1_2337, where the latter can be seen to hold its partner in an embrace, facilitated by hydrophobic and ionic interactions between the proteins. Although Bleg1_2507 shares relatively low sequence identity (47%) to BsSco, interestingly, the predicted metal-binding residues of Bleg1_2507 i.e. Cys-69, Cys-73 and His-159 were located at flexible active loops similar to other Sco proteins across biological taxa. This highlights structural conservation of Sco despite their various functions in prokaryotes and eukaryotes. CONCLUSIONS: We propose that HP Bleg1_2507 is a Sco protein which is able to interact with COXII, its redox partner and therefore, may possess metallochaperone and redox functions similar to other documented bacterial Sco proteins. It is hoped that this scientific effort will help to spur the search for other physiologically relevant proteins among the so-called "orphan" proteins of any given organism.

Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis.

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12.

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 Lichen Flavin-Dependent Halogenase, DnHal: Identification, Heterologous Expression and Functional Characterization.

Enzymatic halogenation captures scientific interest considering its feasibility in modifying compounds for chemical diversity. Currently, majority of flavin-dependent halogenases (F-Hals) were reported from bacterial origin, and as far as we know, none from lichenized fungi. Fungi are well-known producers of halogenated compounds, so using available transcriptomic dataset of Dirinaria sp., we mined for putative gene encoding for F-Hal. Phylogenetic-based classification of the F-Hal family suggested a non-tryptophan F-Hals, similar to other fungal F-Hals, which mainly act on aromatic compounds. However, after the putative halogenase gene from Dirinaria sp., dnhal was codon-optimized, cloned, and expressed in Pichia pastoris, the ~63 kDa purified enzyme showed biocatalytic activity towards tryptophan and an aromatic compound methyl haematommate, which gave the tell-tale isotopic pattern of a chlorinated product at m/z 239.0565 and 241.0552; and m/z 243.0074 and 245.0025, respectively. This study is the start of understanding the complexities of lichenized fungal F-hals and its ability to halogenate tryptophan and other aromatic. compounds which can be used as green alternatives for biocatalysis of halogenated compounds.

Structural prediction of a novel chitinase from the psychrophilic Glaciozyma antarctica PI12 and an analysis of its structural properties and function.

The structure of psychrophilic chitinase (CHI II) from Glaciozyma antarctica PI12 has yet to be studied in detail. Due to its low sequence identity (<30 %), the structural prediction of CHI II is a challenge. A 3D model of CHI II was built by first using a threading approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9v7. Analysis of the catalytic insertion domain structure in CHI II revealed an increase in the number of aromatic residues and longer loops compared to mesophilic and thermophilic chitinases. A molecular dynamics simulation was used to examine the stability of the CHI II structure at 273, 288 and 300 K. Structural analysis of the substrate-binding cleft revealed a few exposed aromatic residues. Substitutions of certain amino acids in the surface and loop regions of CHI II conferred an increased flexibility to the enzyme, allowing for an adaptation to cold temperatures. A substrate binding comparison of CHI II with the mesophilic chitinase from Coccidioides immitis, 1D2K, suggested that the psychrophilic adaptation and catalytic activity at low temperatures were achieved through a reduction in the number of salt bridges, fewer hydrogen bonds and an increase in the exposure of the hydrophobic side chains to the solvent.

Protein surface engineering and interaction studies of maltogenic amylase towards improved enzyme immobilisation.

A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (-7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (-3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20-30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts.

Characterization of AnCUT3, a plastic-degrading paucimannose cutinase from Aspergillus niger expressed in Pichia pastoris.

Cutinases are hydrolytic enzymes secreted by phytopathogens to degrade cutin, the main polymeric component of plant cuticles. The multifaceted functionality of cutinases has allowed for their exploitation for catalytic reactions beyond their natural purpose. To diversify and expand the cutinase enzyme class, we identified five cutinase homologs from the saprotroph Aspergillus niger. One of these cutinases, AnCUT3, was over-expressed in Pichia pastoris and its biophysicochemical properties characterized. The purified recombinant AnCUT3 possessed an optimum temperature of 25 °C, an optimum pH of 5, and was stable at temperatures up to 50 °C (1 h incubation, melting point of 45.6 °C) and in a wide pH range. Kinetic studies of AnCUT3 using pNP ester substrates showed the highest catalytic efficiency, kcat/Km of 859 mM-1 s-1 toward p-nitrophenyl decanoate (C10). Although its calculated molecular mass is 27 kDa, AnCUT3 was expressed as two glycosylated proteins of molecular weights 24 and 50 kDa. Glycan profiling detected the presence of atypical paucimannose N-glycans (≤Man1-5GlcNAc) from recombinant AnCUT3, suggesting protein-dependent glycan processing of AnCUT3 in P. pastoris. AnCUT3 was also able to degrade and modify the surface of polycaprolactone and polyethylene terephthalate. Taken together, these features poise AnCUT3 as a potential biocatalyst for industrial applications.

Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12.

BACKGROUND: Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide ß-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food. RESULTS: A gene encoding a cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE) and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low temperatures. CONCLUSION: By taking advantage of its high activity at low temperatures and its acidic pH optimum, this recombinant chitinase will be valuable in various biotechnological applications under low temperature and acidic pH conditions.

A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli.

L-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS-PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.

An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production.

Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 µM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

Structural and functional characterisation of a cold-active yet heat-tolerant dehydroquinase from Glaciozyma antarctica PI12.

Dehydroquinase or 3-dehydroquinate dehydratase (DHQD) reversibly cleaves 3-dehydroquinate to form 3-dehydroshikimate. Here, we describe the functional and structural features of a cold active type II 3-dehydroquinate dehydratase from the psychrophilic yeast, Glaciozyma antarctica PI12 (GaDHQD). Functional studies showed that the enzyme was active at low temperatures (10-30 °C), but displayed maximal activity at 40 °C. Yet the enzyme was stable over a wide range of temperatures (10-70 °C) and between pH 6.0-10.0 with an optimum pH of 8.0. Interestingly, the enzyme was highly thermo-tolerant, denaturing only at approximately 84 °C. Three-dimensional structure analyses showed that the G. antarctica dehydroquinase (GaDHQD) possesses psychrophilic features in comparison with its mesophilic and thermophilic counterparts such as higher numbers of non-polar residues on the surface, lower numbers of arginine and higher numbers of glycine-residues with lower numbers of hydrophobic interactions. On the other hand, GaDHQD shares some traits (i.e. total number of hydrogen bonds, number of proline residues and overall folding) with its mesophilic and thermophilic counterparts. Combined, these features contribute synergistically towards the enzyme's ability to function at both low and high temperatures.

Entrapment of porous cross-linked enzyme aggregates of maltogenic amylase from Bacillus lehensis G1 into calcium alginate for maltooligosaccharides synthesis.

Maltooligosaccharides (MOSs) are emerging oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). However, the lack of enzyme stability makes this approach unrealistic for industrial applications. The formation of cross-linked enzyme aggregates (CLEAs) is a promising tool for improving enzyme stability, and the substrate accessibility problem of CLEA formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs exhibited high enzyme leaching and low solvent tolerance. To address these problems, p-CLEAs of Mag1 (Mag1-p-CLEAs) were entrapped in calcium alginate beads (CA). Mag1-p-CLEAs-CA prepared with 2.5% (w/v) sodium alginate and 0.6% (w/v) calcium chloride yielded 53.16% (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards beta-cyclodextrin (ß-CD) (Km = 0.62 mM). MOSs (300 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C. Finally, the reusability of Mag1-p-CLEAs-CA makes them as a potential biocatalyst for the continuous synthesis of MOSs.

Functional and structural analyses of an expansin-like protein from the antarctic yeast Glaciozyma antarctica PI12 reveal strategies of nutrient scavenging in the sea ice environment.

Genome data mining of the Antarctic yeast, Glaciozyma antarctica PI12 revealed an expansin-like protein encoding sequence (GaEXLX1). The GaEXLX1 protein is 24.8 kDa with a high alkaline pI of 9.81. Homology modeling of GaEXLX1 showed complete D1 and D2 domains of a conventional expansin. The protein exhibited 36% sequence similarity to Clavibacter michiganensis EXLX1 (PDB: 4JCW). Subsequently, a recombinant GaEXLX1 protein was produced using Escherichia coli expression system. Incubation with Avicel, filter paper and cotton fiber showed that the protein can disrupt the surface of crystalline and pure cellulose, suggesting a cell wall modification activity usually exhibited by expansin-like proteins. Binding assays displayed that GaEXLX1 can bind to polymeric substrates, including those postulated to be present in the sea ice ecosystem such as crab chitin and moss lichenan. GaEXLX1 may assist in the recognition and loosening of these substrates in the sea ice prior to hydrolysis by other extracellular enzymes. Similar loosening mechanism to classical expansin-like protein has been postulated for this psychrophilic protein based on several conserved residues of GaEXLX1 involved in binding interaction identified by docking analyses.

Protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 for catalytic efficiency improvement on kenaf biomass hydrolysis.

Enzyme hydrolysis faces a bottleneck due to the recalcitrance of the lignocellulose biomass. The protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 was performed near the active site and at the N-terminal region to improve its catalytic efficiency towards pretreated kenaf (Hibiscus cannabinus) hydrolysis. Five mutants were constructed by combined approaches of error-prone PCR, site-saturation and site-directed mutagenesis. The double mutant c168 t/Q192H showed the most effective hydrolysis reaction with a 13.9-fold increase in catalytic efficiency, followed by mutants Y7L and c168 t/Q192 H/Y7L with a 1.6-fold increase, respectively. The enhanced catalytic efficiency evoked an increase in sugar yield of up to 28% from pretreated kenaf. In addition, mutant c168 t/Q192 H/Y7L improved the thermostability at higher temperature and acid stability. This finding shows that mutations at distances less than 15 Šfrom the active site and at putative secondary binding sites affect xylanase catalytic efficiency towards insoluble substrates hydrolysis.