Regulation of a novel DsGATA1 from Dunaliella salina on the synthesis of carotenoids under red light.

Dunaliella salina is a high-quality industrial effector for carotenoid production. The mechanism by which red light regulates carotenoid synthesis is still unclear. In this study, a transcription factor of DsGATA1 with a distinct structure was discovered in D. salina. The recognition motif of DsGATA1 was comparable to that of plant and fungal GATA, despite its evolutionary proximity to animal-derived GATA. The expression of DsGATA1 in D. salina was still noticeably decreased when exposed to red light. Analysis of physiological and biochemical transcriptomic data from overexpressed, interfering, and wild-type strains of DsGATA1 revealed that DsGATA1 acts as a global regulator of D. salina carotenoid synthesis. The upregulated genes in the CBP pathway by DsGATA1 were involved in its regulation of the synthesis of carotenoids. DsGATA1 also enhanced carotenoid accumulation under red light by affecting N metabolism. DsGATA1 was found to directly bind to the promoter of nitrate reductase to activate its expression, promoting D. salina nitrate uptake and accelerating biomass accumulation. DsGATA1 affected the expression of the genes encoding GOGAT, GDH, and ammonia transporter proteins. Moreover, our study revealed that the regulation of N metabolism by DsGATA1 led to the production of NO molecules that inhibited carotenoid synthesis. However, DsGATA1 significantly enhanced carotenoid synthesis by NO scavenger removal of NO. The D. salina carotenoid accumulation under red light was elevated by 46% in the presence of overexpression of DsGATA1 and NO scavenger. Nevertheless, our results indicated that DsGATA1 could be an important target for engineering carotenoid production. KEY POINTS: • DsGATA1 with a distinct structure and recognition motif was found in D. salina • DsGATA1 enhanced carotenoid production and biomass in D. salina under red light • DsGATA1 is involved in the regulation of N metabolism and carotenoid synthesis.

Phylogenetic, Structural and Functional Evolution of the LHC Gene Family in Plant Species.

Light-harvesting chlorophyll a/b-binding (LHC) superfamily proteins play a vital role in photosynthesis. Although the physiological and biochemical functions of LHC genes have been well-characterized, the structural evolution and functional differentiation of the products need to be further studied. In this paper, we report the genome-wide identification and phylogenetic analysis of LHC genes in photosynthetic organisms. A total of 1222 non-redundant members of the LHC family were identified from 42 species. According to the phylogenetic clustering of their homologues with Arabidopsis thaliana, they can be divided into four subfamilies. In the subsequent evolution of land plants, a whole-genome replication (WGD) event was the driving force for the evolution and expansion of the LHC superfamily, with its copy numbers rapidly increasing in angiosperms. The selection pressure of photosystem II sub-unit S (PsbS) and ferrochelatase (FCII) families were higher than other subfamilies. In addition, the transcriptional expression profiles of LHC gene family members in different tissues and their expression patterns under exogenous abiotic stress conditions significantly differed, and the LHC genes are highly expressed in mature leaves, which is consistent with the conclusion that LHC is mainly involved in the capture and transmission of light energy in photosynthesis. According to the expression pattern and copy number of LHC genes in land plants, we propose different evolutionary trajectories in this gene family. This study provides a basis for understanding the molecular evolutionary characteristics and evolution patterns of plant LHCs.

Identification of a novel anthocyanin synthesis pathway in the fungus Aspergillus sydowii H-1.

BACKGROUND: Anthocyanins are common substances with many agro-food industrial applications. However, anthocyanins are generally considered to be found only in natural plants. Our previous study isolated and purified the fungus Aspergillus sydowii H-1, which can produce purple pigments during fermentation. To understand the characteristics of this strain, a transcriptomic and metabolomic comparative analysis was performed with A. sydowii H-1 from the second and eighth days of fermentation, which confer different pigment production. RESULTS: We found five anthocyanins with remarkably different production in A. sydowii H-1 on the eighth day of fermentation compared to the second day of fermentation. LC-MS/MS combined with other characteristics of anthocyanins suggested that the purple pigment contained anthocyanins. A total of 28 transcripts related to the anthocyanin biosynthesis pathway was identified in A. sydowii H-1, and almost all of the identified genes displayed high correlations with the metabolome. Among them, the chalcone synthase gene (CHS) and cinnamate-4-hydroxylase gene (C4H) were only found using the de novo assembly method. Interestingly, the best hits of these two genes belonged to plant species. Finally, we also identified 530 lncRNAs in our datasets, and among them, three lncRNAs targeted the genes related to anthocyanin biosynthesis via cis-regulation, which provided clues for understanding the underlying mechanism of anthocyanin production in fungi. CONCLUSION: We first reported that anthocyanin can be produced in fungus, A. sydowii H-1. Totally, 31 candidate transcripts were identified involved in anthocyanin biosynthesis, in which CHS and C4H, known as the key genes in anthocyanin biosynthesis, were only found in strain H1, which indicated that these two genes may contribute to anthocyanins producing in H-1. This discovery expanded our knowledges of the biosynthesis of anthocyanins and provided a direction for the production of anthocyanin.

Purification and characterization of a novel intracellular α-amylase with a wide variety of substrates hydrolysis and transglycosylation activity from Paenibacillus sp. SSG-1.

Intracellular α-amylase was a special glycoside hydrolase in the cytoplasm. We cloned and expressed an intracellular α-amylase, Amy, from Paenibacillus sp. SSG-1. The recombinant enzyme was purified by metal-affinity chromatography, exhibited a molecular mass of 71.7 kDa. Amy exhibited unexpectedly sequence similarity and evolutionary relationships with alpha-glucanotransferase. The docked results of Amy with maltose showed it had similar catalytic residues with α-amylase and glucanotransferase. The substrate specificity experiment showed that Amy could hydrolyze typical substrates into glucose and maltose. It was noteworthy that Amy showed the catalytic capacity of cyclomaltodextrinase and pullulanase. Meanwhile, Amy could transfer sugar molecules and form maltotetraose upon the hydrolysis of substrates. These results indicated that Amy was a novel intracellular α-amylase with distinct catalytic ability characteristics of hydrolyzing glycogen/cyclodextrin/pullulan and transglycosylation. We deduced that Amy may play an important role in utilizing maltooligosaccharides that released from extracellular α-glucan or storage α-glucan (glycogen) in Paenibacillus sp. SSG-1.

Chromium removal from solution by five photosynthetic bacteria isolates.

Biological method has been recognized as a low-cost and ecofriendly approach for removing heavy metals from aqueous wastes. In this study, the ability of five photosynthetic bacteria isolates (strains labeled SC01, HN02, SC05, JS01, and YN01) was examined for their ability to remove Cr from Cr-containing solutions. Furthermore, the possible removal mechanisms were elucidated by comparing chromium removal rates, antioxidant reaction, and accumulation of reactive oxygen species (ROS). Among the five bacteria, strains SC01 and SC05 presented the highest removal rates of chromium ions and the activity of cysteine desulfhydrase under Cr stress. They also showed lower levels of ROS and cell death than the other three bacteria strains under Cr stress. In addition, total bacteriochlorophyll content and activities of six antioxidant enzymes in SC01 were highest among these selected strains. On the contrary, strain HN02 presented the lowest level of Cr removal and the lowest activities of antioxidant enzymes. It also exhibited the highest level of ROS under Cr(VI) stress. Overall, these results show that the strains SC01 and SC05 have good Cr removal ability and could be used for removal of Cr in industrial effluents.

Isolation and characterization of agar-degrading endophytic bacteria from plants.

Agar is a polysaccharide extracted from the cell walls of some macro-algaes. Among the reported agarases, most of them come from marine environment. In order to better understand different sources of agarases, it is important to search new non-marine native ones. In this study, seven agar-degrading bacteria were first isolated from the tissues of plants, belonging to three genera, i.e., Paenibacillus sp., Pseudomonas sp., and Klebsiella sp. Among them, the genus Klebsiella was first reported to have agarolytic ability and the genus Pseudomonas was first isolated from non-marine environment with agarase activity. Besides, seven strains were characterized by investigating the growth and agarase production in the presence of various polysaccharides. The results showed that they could grow on several polysaccharides such as araban, carrageenan, chitin, starch, and xylan. Besides, they could also produce agarase in the presence of different polysaccharides other than agar. Extracellular agarases from seven strains were further analyzed by SDS-PAGE combined with activity staining and estimated to be 75 kDa which has great difference from most reported agarases.

Molecular cloning and functional characterization of MnSOD from Dunaliella salina.

Dunaliella salina, a unicellular green alga, has the potential to grow in hypersaline environments via one of its gene products, superoxide dismutase (SOD). The superoxide radicals (O2 (-) ) produced by environmental stresses can cause damage to cells, and SOD catalyzes the turnover of such free radicals to protect cells. In this study, the gene coding for SOD in D. salina was cloned and the product was further identified and characterized. The open reading frame of this gene was 651 bp long, encoding for 217 amino acids. According to the sequence alignment using BLAST, native polyacrylamide electrophoresis for SOD activity analysis, and atomic absorption spectroscopy analysis, this protein belongs to the manganese-containing superoxide dismutase (MnSOD) family. Complementation analysis, performed by introducing plasmids carrying an inducible version of the D. salina gene encoding for MnSOD into an SOD-deficient mutant of E.coli, revealed that this gene could not only complement the defects in SOD activity, but was also capable of providing a stronger tolerance to restrictive growth conditions, such as high salt and prolonged UV exposure, compared to the tolerance of wild-type strains.

Fluorimetric analysis of the binding characteristics of 5-enolpyruvylshikimate-3-phosphate synthase with substrates in Dunaliella salina.

A general model of the catalytic mechanism for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) has already been proposed. But whether shikimate-3-phosphate (S3P) alone can cause EPSPs' conformation changes, and whether the binding site of phosphoenolpyruvate (PEP) and glyphosate is the same are still in debate. In this paper, DsaroA gene amplified and cloned from Dunaliella salina (our laboratory's early study) was used for DsEPSPs expression and purification. Then the DsEPSP conformation changes as it bind with different substrates were detected by fluorimetry. The results show that we obtained the DsEPSPs by prokaryotic expression and purification, and the S3P binding with DsEPSPs alone cannot cause DsEPSPs to form "close" conformation directly. However, when S3P exits, DsEPSPs did have a trend to change to the "close" conformation. Then the "close" conformation can be formed completely with the addition of phosphoenolpyruvate (PEP) or glyphosate. The inorganic phosphorus can help S3P to induce two domains of DsEPSPs to form "close" conformation. Besides, when DsEPSPs binds with S3P, in 295 nm, only the intensity of emission peak decreases, however, in 280 nm, not only the peak intensity reduces but also the blue-shift phenomenon takes place. The reason for blue-shift phenomenon was the distribution of aromatic amino acids in EPSPs. EPSPs is a good target for novel antibiotics and herbicides, because of shikimic acid pathway is only present in plants and microorganisms, completely absent in mammals, fish, birds, reptiles, and insects. The results demonstrate that the binding of substrates to EPSPs causes a conformational change from an open form to a closed form, that might be important for designing of novel antimicrobial and herbicidal agents that block closure of the enzyme.

PCR-RFLP analysis of the diversity of phytate-degrading bacteria in the Tibetan Plateau.

Phytases play a very important role in increasing phytate digestion and reducing phosphorus pollution in the environment, and phytate-degrading bacteria have a ubiquitous distribution in the environment. Due to its extremely harsh environment, the Tibetan Plateau breeds possibly abundant, extreme microorganisms. In this research, 67 phytate-degrading bacteria were isolated from different habitats in the Tibetan Plateau. Among all isolates, 40.3% were screened from farmland, 25.3% from wetland, 4.5% from saline-alkaline soil, 7.5% from hot springs, and 22.4% from lawns, which showed that the distribution of the phytate-degrading bacteria varied with habitats. By the PCR-RFLP method, 16 different species were identified and named, 4 of which are reported for the first time as phytate-degrading bacteria, that is, Uncultured Enterococcus sp. GYPB01, Bacillaceae bacterium strain GYPB05, Endophytic bacterium strain GYPB16, and Shigella dysenteria strain GYPB22. Through the assay of phytase activity of 16 strains, Klebsiella sp. strain GYPB15 displayed the highest capability of phytase production. Through analysis of the optimum pH, the optimum temperature, and the thermal stability of enzyme from 16 strains, some especial phytate-degrading bacteria were obtained. Our findings clearly indicate a good relation between the composition of the soils from the different environments in the Tibetan Plateau and populations of cultivable phytate-degrading bacteria. Moreover, extreme harsh soils are logically the best soils in which to find some strains of phytate-degrading bacteria for exploiting in the fields of biotechnology and industry.

AppA C-terminal plays an important role in its thermostability in Escherichia coli.

Due to our previous research, mainly the thermostable mutants Q307D, Y311K, and I427L, we conjectured that Escherichia coli AppA phytase's C-terminal plays an important role in its thermostability, and AppA begins to collapse from the C-terminal when at a higher temperature. So here we constructed C-lose mutant to prove it. The residual activities of the wild-type AppA phytase and C-lose were 31.42 and 70.49 %, respectively, after being heated at 80 °C for 10 min. The C-terminal deletion mutant C-lose showed 39.07 % thermostability enhancement than the wild-type both without the pH and temperature optimum changed. It proved the C-lose plays a key role in E. coli AppA phytase's thermostability.

A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli AppA phytase.

Despite recent advances in our understanding of the importance of protein surface properties for protein thermostability,there are seldom studies on multi-factors rational design strategy, so a more scientific, simple and effective rational strategy is urgent for protein engineering. Here, we first attempted to use a three-factors rational design strategy combining three common structural features, protein flexibility, protein surface, and salt bridges. Escherichia coli AppA phytase was used as a model enzyme to improve its thermostability. Moreover, the structure and enzyme features of the thermostable mutants designed by our strategy were analyzed roundly. For the single mutants, two (Q206E and Y311K), in five exhibited thermostable property with a higher success rate of prediction (40 %). For the multiple mutants, the themostable sites were combined with another site, I427L, we obtained by directed evolution, Q206E/I427L, Y311K/I427L, and Q206E/Y311K/I427L, all exhibited thermostable property. The Y311K/I427L doubled thermostability (61.7 %, and was compared to 30.97 % after being heated at 80 °C for 10 min) and catalytic efficiency (4.46 was compared to 2.37) improved more than the wild-type AppA phytase almost without hampering catalytic activity. These multi-factors of rational design strategy can be applied practically as a thermostabilization strategy instead of the conventional single-factor approach.

Multi-drug resistant gram-negative enteric bacteria isolated from flies at Chengdu Airport, China.

We collected flies from Chengdu Shuangliu International Airport to examine for the presence of bacteria and to determine the sensitivity patterns of those bacteria. A total of 1,228 flies were collected from 6 sites around Chengdu Shuangliu International Airport from April to September 2011. The predominant species was Chrysomya megacephala (n=276, 22.5%). Antimicrobial-resistant gram-negative enteric bacteria (n=48) were isolated from flies using MacConkey agar supplemented with cephalothin (20 microg/ml). These were identified as Escherichia coli (n=37), Klebsiella pneumoniae (n=6), Pseudomonas aeruginosa (n=3) and Aeromonas hydrophila (n=2). All isolated bacteria were tested for resistance to 21 commonly used antimicrobials: amoxicillin (100%), ticarcillin (100%), cephalothin (100%), cefuroxime (100%), ceftazidime 1 (93.8%), piperacillin (93.8%), cefotaxime (89.6%), ticarcillin-clavulanate (81.3%), trimethoprim-sulfamethoxazole (62.5%), ciprofloxacin (54.2%), gentamicin (45.8%), cefepime (39.6%), tobramycin (39.6%), ceftazidime (22.9%), cefoxitin (16.7%), amikacin (16.7%), netilmicin (14.6%), amoxicillin-clavulanate (6.3%) and piperacillin-tazobactam (2.1%). No resistance to meropenem or imipenem was observed. Antibiotic resistance genes among the isolated bacteria were analyzed for by polymerase chain reaction. Thirty of the 48 bacteria with resistance (62.5%) possessed the blaTEM gene.

Distribution, characterization, and evolution of heavy metal resistance genes and Tn7-like associated heavy metal resistance Gene Island of Burkholderia.

Introduction: Burkholderia is a rod-shaped aerobic Gram-negative bacteria with considerable genetic and metabolic diversity, which can beused for bioremediation and production applications, and has great biotechnology potential. However, there are few studies on the heavy metal resistance of the Burkholderia genus. Methods: In this paper, the distribution, characteristics and evolution of heavy metal resistance genes in Burkholderia and the gene island of Tn7-like transposable element associated with heavy metal resistance genes in Burkholderia were studied by comparative genomic method based on the characteristics of heavy metal resistance. Results and discussion: The classification status of some species of the Burkholderia genus was improved, and it was found that Burkholderia dabaoshanensis and Burkholderia novacaledonica do not belong to the Burkholderia genus.Secondly, comparative genomics studies and pan-genome analysis found that the core genome of Burkholderia has alarger proportion of heavy metal resistance genes and a greater variety of heavy metalresistance genes than the subsidiary genome and strain specific genes. Heavy metal resistance genes are mostly distributed in the genome in the form of various gene clusters (for example, mer clusters, ars clusters, czc/cusABC clusters). At the same time, transposase, recombinase, integrase and other genes were foundupstream and downstream of heavy metal gene clusters, indicating that heavy metal resistance genes may beobtained through horizontal transfer. The analysis of natural selection pressure of heavy metal resistance genes showed that heavy metal resistance genes experienced strong purification selection under purification selection pressure in the genome.The Tn7 like transposable element of Burkholderia was associated with the heavy metal resistance gene island, and there were a large number of Tn7 transposable element insertion events in genomes. At the same time, BGI metal gene islands related to heavy metal resistance genes of Tn7 like transposable element were found, and these gene islands were only distributed in Burkholderia cepacia, Burkholderia polyvora, and Burkholderia contaminant.

Molecular characterization and expression profiling of two flavohemoglobin genes play essential roles in dissolved oxygen and NO stress in Saitozyma podzolica zwy2-3.

Flavohemoglobins (Fhbs) are key enzymes involved in microbial nitrosative stress resistance and nitric oxide degradation. However, the roles of Fhbs in fungi remain largely unknown. In this study, SpFhb1 and SpFhb2, two flavohemoglobin-encoding genes in Saitozyma podzolica zwy2-3 were characterized. Protein structure analysis and molecular docking showed that SpFhbs were conserved in bacteria and fungi. Phylogenetic analysis revealed that SpFhb2 may be acquired through the transfer event of independent horizontal genes from bacteria. The expression levels of SpFhb1 and SpFhb2 showed opposite trend under high/low dissolved oxygen, implying that they may exhibited different functions. Through deletion and overexpression of SpFhbs, we confirmed that SpFhbs were conducive to lipid accumulation under high stress. The sensitivities of ΔFhb mutants to NO stress were significantly increased compared with that in the WT, indicating that they were required for NO detoxification and nitrosative stress resistance in S. podzolica zwy2-3. Furthermore, SpAsg1 was identified that simultaneously regulates SpFhbs, which functions in the lipid accumulation under high/low dissolved oxygen and NO stress in S. podzolica zwy2-3. Overall, two different SpFhbs were identified in this study, providing new insights into the mechanism of lipid accumulation in fungi under high/low dissolved oxygen and NO stress.

Potential xylose transporters regulated by CreA improved lipid yield and furfural tolerance in oleaginous yeast Saitozyma podzolica zwy-2-3.

Lignocellulose's hydrolysate, a significant renewable source, contains xylose and furfural, making it challenging for industrial production of oleaginous yeast. On xylose fermentation with furfural treatment, OE::DN7263 and OE::DN7661 increased lipid yield and furfural tolerance versus WT, while, which of OE::CreA were decreased owing to CreA regulating DN7263 and DN7661 negatively. OE::CreA generated reactive oxygen species (ROS) causing oxidative damage. OE::DN7263, OE::DN7661, and ΔCreA reduced furfural via NADH; while ΔCreA produced less ROS and OE::DN7263, and OE::DN7661 scavenged ROS quickly, minimizing oxidative damage. Overall, CreA knockout increased DN7263 and DN7661 expression to facilitate xylose assimilation, enhancing NADH generation and ROS clearance. Finally, with mixed sugar fermentation, ΔCreA and OE::DN7263's biomass and lipid yield rose without furfural addition, while that of ΔCreA remained higher than WT after furfural treatment. These findings revealed how oleaginous yeast zwy-2-3 resisted furfural stress and indicated ΔCreA and OE::DN7263 might develop into robust industrial chassis strains.

GATA-type transcriptional factor SpGAT1 interacts with SpMIG1 and promotes lipid accumulation in the oleaginous yeast [Formula: see text] zwy-2-3.

BACKGROUND: In oleaginous yeast, nitrogen limitation is a critical parameter for lipid synthesis. GATA-family transcriptional factor GAT1, a member of the target of rapamycin (TOR) pathway and nitrogen catabolite repression (NCR), regulates nitrogen uptake and utilization. Therefore, it is significant to study the SpGAT1 regulatory mechanism of lipid metabolism for conversion of biomass to microbial oil in [Formula: see text] zwy-2-3. RESULTS: Compared with WT, [Formula: see text], and OE::gat1, the lipid yield of OE::gat1 increased markedly in the low carbon and nitrogen ratio (C/N ratio) mediums, while the lipid yield and residual sugar of [Formula: see text] decreased in the high C/N ratio medium. According to yeast two-hybrid assays, SpGAT1 interacted with SpMIG1, and its deletion drastically lowered SpMIG1 expression on the high C/N ratio medium. MIG1 deletion has been found in earlier research to affect glucose metabolic capacity, resulting in a prolonged lag period. Therefore, we speculated that SpGAT1 influenced glucose consumption rate across SpMIG1. Based on yeast one-hybrid assays and qRT-PCR analyses, SpGAT1 regulated the glyoxylate cycle genes ICL1, ICL2, and pyruvate bypass pathway gene ACS, irrespective of the C/N ratio. SpGAT1 also could bind to the ACAT2 promoter in the low C/N medium and induce sterol ester (SE) accumulation. CONCLUSION: Our findings indicated that SpGAT1 positively regulated lipid metabolism in S.podzolica zwy-2-3, but that its regulatory patterns varied depending on the C/N ratio. When the C/N ratio was high, SpGAT1 interacted with SpMIG1 to affect carbon absorption and utilization. SpGAT1 also stimulated lipid accumulation by regulating essential lipid anabolism genes. Our insights might spur more research into how nitrogen and carbon metabolism interact to regulate lipid metabolism.

DsLCYB Directionally Modulated ß-Carotene of the Green Alga Dunaliella salina under Red Light Stress.

Carotenoids, which are natural pigments found abundantly in wide-ranging species, have diverse functions and high industrial potential. The carotenoid biosynthesis pathway is very complex and has multiple branches, while the accumulation of certain metabolites often affects other metabolites in this pathway. The DsLCYB gene that encodes lycopene cyclase was selected in this study to evaluate ß-carotene production and the accumulation of ß-carotene in the alga Dunaliella salina. Compared with the wild type, the transgenic algal species overexpressed the DsLCYB gene, resulting in a significant enhancement of the total carotenoid content, with the total amount reaching 8.46 mg/g for an increase of up to 1.26-fold. Interestingly, the production of α-carotene in the transformant was not significantly reduced. This result indicated that the regulation of DsLCYB on the metabolic flux distribution of carotenoid biosynthesis is directional. Moreover, the effects of different light-quality conditions on ß-carotene production in D. salina strains were investigated. The results showed that the carotenoid components of ß-carotene and ß-cryptoxanthin were 1.8-fold and 1.23-fold higher than that in the wild type under red light stress, respectively. This suggests that the accumulation of ß-carotene under red light conditions is potentially more profitable.

Role of Lys281 in the Dunaliella salina (6-4) photolyase reaction.

His(354) and His(358), two highly conserved histidines in Xenopus laevis (6-4) photolyase [equivalent to His(401) and His(405), in Dunaliella salina (6-4) photolyase], are critical for photoreactivation. They act as a base and an acid, respectively. However, the remaining high repair activity when the pH value is higher than the pKa of histidine suggests the involvement of other basic amino acids in photoreactivation. According to the results of in vivo enzyme assay and three-dimension structural model of Dunaliella salina (6-4) photolyase we hypothesized that Lys(281) might be involved in the photoreactivation over the pH range from 10.0 to 11.0. To test this, we generated two mutant forms of the (6-4) photolyase, K281G and K281R mutant, by overlap extension polymerase chain reaction, and performed the enzyme assay with these mutants. From these results we conclude that the Lys(281), which is highly conserved in (6-4) photolyases, participates in the photoreactivation and acts as an acid to donate a proton to His(401) when the environmental pH is higher than the pKa value of histidine.

Site-directed mutagenesis and thermostability of xylanase XYNB from Aspergillus niger 400264.

Xylanase is one of the most important hemicellulases in industry. However, its low thermostability limits its applications. In this study, one thermostable xylanase-producing strain 400264 was obtained from screening 11 Aspergillus niger strains (producing thermotolerant xylanase), and the optimum temperature of crude xylanase extracted from it was 55°C. Original activity of the crude xylanase is 64% at 60°C and 55% at 85°C with an incubation time of 30 min, respectively. After the expression of recombinant xylanase gene (xynA/xynB), the XYNB (xylanase B) showed higher thermostability than XYNA (xylanase A). Recombinant enzyme XYNB retained 94% of its activity for 10 min at 85°C, while XYNA with no activity left. Site-directed mutagenesis was performed to replace Ala33 of XYNB by Ser33 resulting 19% decrease in enzyme activity after incubating at 85°C for 30 min. It suggested that the Ala33 residue may have a certain effect on the thermophilic adaptation of xylanase.

Modifying thermostability of appA from Escherichia coli.

In order to improve the thermostability of Escherichia coli AppA phytase, Error-prone PCR was used to randomize mutagenesis appA gene, and a gene mutation library was constructed. A mutant I408L was selected from the library by the method of high-throughput screening with 4-methyl-umbelliferylphosphate (4-MUP). The appA gene of the mutant was cloned and expressed in E. coli Origami (DE3). The recombinant protein was purified by Ni-affinity chromatography, and the enzymatic features were analyzed. The results indicated that AppA phytase activities of mutant I408L and wild-type (WT) strain remained at 51.3 and 28%, respectively, after treatment at 85°C for 5 min. It means that the thermostability enhancement of AppA phytase I408L was 23.3% more as compared with WT. The K (m) of both phytase were 0.18 and 0.25 mM, respectively, which indicated that the catalyzing efficiency of I408L was improved. AppA phytase of mutant I408L showed a significant enhancement against trypsin, which was nearly three times compared with WT. In addition, AppA phytase of mutant could be activated by Mg(2+) and Mn(2+); in contrast, it could be inhibited by Ca(2+), Co(2+), Cu(2+), and K(+) in varying degrees, and the enzymatic activity was almost lost the presence of Fe(3+) and Zn(2+). It appears that screening thermotolerant phytase of E. coli by high throughput screening with a fluorescence substrate is a fast, simple, and effective method. The mutant I408L obtained in this study could be used for the large-scale commercial production of phytase.