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Chlorophyllide (chlide) is a natural catabolic product of chlorophyll (Chl), produced through the activity of chlorophyllase (chlase). The growth inhibitory and antioxidant effects of chlide from different plant leaf extracts have not been reported. The aim of this study is to demonstrate that chlide in crude extracts from leaves has the potential to exert cytotoxic effects on cancer cell lines. The potential inhibitory and antioxidant effects of chlide in crude extracts from 10 plant leaves on breast cancer cells (MCF7 and MDA-MB-231), hepatocellular carcinoma cells (Hep G2), colorectal adenocarcinoma cells (Caco2), and glioblastoma cells (U-118 MG) were studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and DPPH (1,1-diphenyl-2-picrylhydrazyl) assays. The results of the MTT assay showed that chlide in crude extracts from sweet potato were the most effective against all cancer cell lines tested. U-118 MG cells were the most sensitive, while Caco2 cells were the most resistant to the tested crude extracts. The cytotoxic effects of chlide and Chl in crude extracts from sweet potato and of commercial chlorophyllin (Cu-chlin), in descending order, were as follows: chlide > Chl > Cu-chlin. Notably, the IC50 of sweet potato in U-118 MG cells was 45.65 μg/mL while those of Chl and Cu-chlin exceeded 200 μg/mL. In the DPPH assay, low concentrations (100 μg/mL) of chlide and Cu-chlin from crude extracts of sweet potato presented very similar radical scavenging activity to vitamin B2. The concentration of chlide was negatively correlated with DPPH activity. The current study was the first to demonstrate that chlide in crude extracts from leaves have potential cytotoxicity in cancer cell lines. Synergism between chlide and other compounds from leaf crude extracts may contribute to its cytotoxicity.

Astaxanthin possesses various biological properties and is used in the animal and fish feed, food, and beverage industries. In this study, we derived zeaxanthin biosynthesis genes (crtE, crtB, crtI, crtY, and crtZ) from Erwinia uredovora and crtW from Agrobacterium aurantiacum. We fused inducible and constitutive promoters to astaxanthin biosynthesis genes to construct a novel plasmid (dubbed PTP3-6) that can effectively enhance free-form astaxanthin (FFAX) production. The PTP3-6 plasmid contains one T7 promoter, driving IPTG inducible crtW expression, and three constitutive promoters (isolated from E. uredovora) driving expression of the other zeaxanthin biosynthesis genes. Escherichia coli BL21 (DE3) cells carrying the PTP3-6 plasmid produced 8.3 mg/g dry cell weight astaxanthin, which is 69.4-fold higher than has been previously reported. Using multiple promoter fusions of astaxanthin biosynthesis genes could be applied in other hosts to enhance astaxanthin production. FFAX was identified in recombinant E. coli cells through ultra-performance liquid chromatography-mass spectrometry.

The fungus Antrodia cinnamomea has been used as a folk medicine for various diseases, especially cancer. When A. cinnamomea is cultured on the original host, an endangered woody plant Cinnamomum kanehirai Hayata, the fungus produces more active ingredients, but its growth is slow. Here, C. kanehirai leaf ethanol extract (KLEE) was used as a substitute for C. kanehirai wood to culture A. cinnamomea on solid medium to shorten the culture period and produce active metabolites en masse. The antioxidant activities of methanol extracts from A. cinnamomea cultured on KLEE (MEAC-KLEE) were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, reducing power, and ferrous ion-chelating effect, and the effective concentration (EC50) values were 0.27, 0.74, and 0.37 mg mL−1, respectively. MEAC-KLEE exhibited specific anti-proliferative activity against a non-small-cell lung cancer cell line (A549) by Annexin V assay. A secondary metabolite (2,4-dimethoxy-6-methylbenzene-1,3-diol, DMMB) present in the extract (MEAC-KLEE) was purified by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) spectra. DMMB exhibited moderate antioxidant activity against DPPH radicals and reducing power, with EC50 values of 12.97 and 25.59 μg mL−1, respectively, and also induced apoptosis in A549 cells. Our results provide valuable insight into the development of DMMB for nutraceutical biotechnology.

Chlorophyllides can be found in photosynthetic organisms. Generally, chlorophyllides have a-, b-, c-, d-, and f-type derivatives, and all chlorophyllides have a tetrapyrrole structure with a Mg ion at the center and a fifth isocyclic pentanone. Chlorophyllide a can be synthesized from protochlorophyllide a, divinyl chlorophyllide a, or chlorophyll. In addition, chlorophyllide a can be transformed into chlorophyllide b, chlorophyllide d, or chlorophyllide f. Chlorophyllide c can be synthesized from protochlorophyllide a or divinyl protochlorophyllide a. Chlorophyllides have been extensively used in food, medicine, and pharmaceutical applications. Furthermore, chlorophyllides exhibit many biological activities, such as anti-growth, antimicrobial, antiviral, antipathogenic, and antiproliferative activity. The photosensitivity of chlorophyllides that is applied in mercury electrodes and sensors were discussed. This article is the first detailed review dedicated specifically to chlorophyllides. Thus, this review aims to describe the definition of chlorophyllides, biosynthetic routes of chlorophyllides, purification of chlorophyllides, and applications of chlorophyllides.

Antodia cinnamomea, a precious brown-rot fungus endemic to Taiwan, has pharmaceutical applications due to its diverse array of metabolites. The terpenoids found in A. cinnamomea contribute to its most important bioactivities. We identified several terpenoid compounds in A. cinnamomea and revealed that their content in mycelium and fruiting body were significantly different. Using next-generation sequencing and an in-house transcriptome database, we identified several terpene synthase (TPS) candidates. After sequence analysis and functional characterization, 10 out of 12 candidates were found to have single or multiple terpene synthesis functions. Most of the terpenoid compounds were found to confer important bioactivities. RT-PCR results showed a positive correlation between terpene synthase expression pattern and terpenoid content. In addition, we identified several modification enzyme candidates that may be involved in the postmodification of terpenoid compounds with a genomic DNA scaffold, and a putative genetic network.

Esterases are widely used in the food industry. Here, a new thermophilic bacterium, Geobacillus thermodenitrificans PS01, was isolated and the esterase-encoding gene est1 was cloned, sequenced, and recombinant expressed in Escherichia coli Tuner (DE3). The highest activity of recombinant Est1 was detected at pH 8.0, and 40 °C and the extreme stability was observed at pH 6–9 over 30 days at 4 °C. In particular, Est1 can hydrolyze short- to medium-chain (C2–C10) triglycerides and p-nitrophenyl esters (C2–C12) and was not inhibited by most metal ions. Kinetic parameters of p-nitrophenyl butyrate hydrolysis under optimal conditions were determined: Km, 22.76 μM; kcat, 10,415 s−1; and kcat/Km, 457.53 μM−1 s−1. The outstanding specification of Est1 indicates its potential for use in industrial applications.

Chlorophyll is a valuable bioactive compound, which is used as a natural food coloring agent and a photosensitizer for photodynamic therapy because of its antioxidant properties, antimutagenic ability, and near-infrared fluorescence. However, chlorophyll is unstable when it comes to retaining its antioxidant activity, when exposed to oxygen, high temperature, or light environments. To enhance the stability of chlorophyll, a polymer encapsulation method was proposed. Polycaprolactone (PCL) was employed to encapsulate the chlorophyll, and the particles size of the composites was controlled through droplet microfluidics. The composites (chlorophyll-encapsulated PCL particles) were characterized through UV–VIS spectrometry, SEM, optical microscopy, and light exposure. The particles were spherical, with diameters adjustable from 68 to 247 μm. Additionally, the chlorophyll-encapsulated PCL particles exhibited considerably prolonged chlorophyll stability. The solid microparticle is more convenient for storage and transportation, and have great potential for application in the food industry.

The purity of chlorophylls plays one of the key role for the production of chlorophyllides. We have designed a facile method for chlorophyll purification by twice solvent extraction. Twice extraction causes the loss of chlorophylls, but the purity of total chlorophylls can be enhanced 182%. Then, the purified chlorophylls can be converted to relatively pure chlorophyllides facilely. The results show that higher purity of chlorophyllides could be obtained when purified chlorophylls (ethanol-hexane extract) was used as starting materials than that of crude chlorophylls (ethanol-only extract). In biocompatibility test, the results showed that the prepared chlorophyllides can be applied as biomaterials. When the prepared chlorophyllides were applied to anticancer tests, they were active both in MCF7 and MDA-MB-231 (multidrug resistant breast cancer cells) cell lines. In addition, the results suggested that the prepared chlorophyllides could be a potential candidate of combination therapy with doxorubicin to breast cancers.

The yeast Candida rugosa can secrete a mixture of lipase isoenzymes (Lips), which have been widely applied in industry. Eight Lip genes (LIP1 to LIP8) have been identified and are expressed in Pichia pastoris. However, the expression level was not sufficient for economical industrial application. In this study, two combined processes of antibiotic selection and low-temperature culture efficiently elicited a high-level secretion of recombinant Lip2 in P. pastoris. The LIP2 gene copy number of the Pichia transformants was increased by sequential selections at gradually increasing Zeocin concentrations. After the first selection at 500 μg/mL of Zeocin, three clones (500-clones) with 2.4-fold to 5.8-fold improvement in Lip2 secretion were identified from 105 survival clones through lipase activity screening. Although the maximum number of LIP2 gene copy was four among these three 500-clones, the lipase secretion of the four-copy clone was not higher than that of the three-copy clone. The effects of multiple gene copy number and low culture temperature resulted in a maximal 32-fold increase in Lip2 secretion. This method could be applied to other Lip isoforms to enhance their yields in P. pastoris.

The Staphylococcus epidermidis lipase (SeLip, GehC) can be used in flavour-compound production via esterification in aqueous solution. This study reports the crystallization and crystallographic analysis of recombinant GehC (rGehC; Lys303–Lys688) with a molecular weight of 43 kDa. rGehC was crystallized at 293 K using PEG 10 000 as a precipitant, and a 99.9% complete native data set was collected from a cooled crystal at 77 K to a resolution of 1.9 Å with an overall R merge value of 7.3%. The crystals were orthorhombic and belonged to space group P212121, with unit-cell parameters a = 42.07, b = 59.31, c = 171.30 Å, α = β = γ = 90°. Solvent-content calculations suggest that there is likely to be one lipase subunit in the asymmetric unit.

Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 °C, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce chlorophyllide derivatives.

The lacI gene in the plasmid bearing the T7A1 promoter ( P A1 )-driven lacZ was randomly mutated. The mutant library was then screened in Escherichia coli B strain deficient in lacI and lacZ . Based on the LacZ phenotype, one heat-sensitive lacI ( lacI ts) was isolated and it revealed a mutation with an amino acid substitution, Met42Lys (designated lacI42 ts). To examine its performance, the lacI42 ts/ P A1 -based plasmid was employed for expression of gehC (encoding lipase) in E. coli B strain. Consequently, the strain that received a thermal induction produced 49-fold more GehC in terms of activity than the uninduced level. The expression condition was further optimized, finally leading to a 47% increase in the GehC activity for the strain. Overall, it indicates that the thermo-regulated vector is useful for the recombinant protein production in E. coli B strain.

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

Bacteriopheophorbide a (BPheid a) is used as a precursor for bacteriochlorin a (BCA), which can be used for photodynamic therapy in both in vitro and in vivo biochemical applications. This study successfully isolated and expressed a photosynthetic bacterium (Cyanothece sp. ATCC 51142) chlorophyllase called CyanoCLH, which can be used as a biocatalyst for the production of a BCA precursor by degrading bacteriochlorophyll a (BChl a). Substrate specificity and enzyme kinetic analyses were performed and the results verified that the recombinant CyanoCLH preferred hydrolyzing BChl a to produce bacteriochlorophyllide a (BChlide a), which can be converted to BPheid a by removing magnesium ion. The recombinant CyanoCLH was cloned and expressed in Escherichia coli BL-21 (DE3), and its molecular weight was 54.7 kDa. The deduced amino acid sequence of the recombinant CyanoCLH comprised a unique lipase-motif GHSLG, which differs from the GHSRG sequence of other plants and lacks a histidine of the typical and conserved catalytic triad Ser-Asp-His. The recombinant CyanoCLH was subjected to biochemical analyses, and the results indicated that its optimal pH and temperature were 7.0 and 60 °C, respectively.

To facilitate the purification of trehalose, an enzymatic process for the selective conversion of glucose was investigated. An epoxy-activated acrylic matrix was used for the immobilization of glucose oxidase. Due to the relatively hydrophobic nature of carrier surface, significant enhancement in enzyme load was observed in the presence of 1000 mM phosphate without observable enzyme denaturation. Upon immobilization glucose oxidase, with optimal activity at 40 °C and pH 7.0, was shown to have higher residual activity at elevated pHs and temperatures. In repeated-batch operations, the immobilized glucose oxidase, with a catalytic activity of 214.06 ± 5.34 U/g gel at an enzyme load of 11.31 ± 0.19 mg/g gel, exhibited sound operation stability for up to 12 cycles, beyond that the activity declined steadily, due to the sequential inactivation of catalase and glucose oxidase by the accumulated hydrogen peroxide. It was shown that the inactivation of enzymes can be alleviated by the addition of hydrogen peroxide scavengers. It was also shown that the gluconic acid thus formed can be readily adsorbed with an ion exchanger leaving trehalose in the solution. The results obtained in this study demonstrate that the proposed process could facilitate the purification of trehalose enzymatically converted from maltose.

Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyllide and phytol.

In this present study, we introduce a fundamental framework and provide information regarding the possible roles of GDSL-type esterase/lipase gene family in Arabidopsis. GDSL-type esterases/lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity, regiospecificity, and stereoselectivity. In this study, we identified 105 GDSL-type esterase/lipase genes in Arabidopsis thaliana by conducting a comprehensive computational analysis. Expression studies indicated that GDSL-type lipase proteins showed varied expression patterns. Phylogenetic tree analysis indicated that AtGELP (Arabidopsis thaliana GDSL-type esterase/lipase protein) gene family was divided into four clades. The phylogenetic analysis, combined with protein motif architectures, and expression profiling were used to predict the roles AtGELP genes. To investigate the physical roles of the AtGELP gene family, we successfully screened 88 AtGELP T-DNA knockout lines for 54 AtGELP genes from 199 putative SALK T-DNA mutants. Transgenic plants of AtGELP genes were used to elucidate the phenotypic characteristics in various developmental stages or stress conditions. Our results suggest that the AtGELP genes have diverse physical functions such as affecting the germination rate and early growth of seedlings subjected to high concentrations of glucose, or being involved in biotic stress responses.

This paper demonstrates a simple and easy approach for the one-step synthesis of Fe₃O₄-chitosan composite particles with tadpole-like shape. The length and diameter of the particles were adjustable from 638.3 μm to ca. 798 μm (length), and from 290 μm to 412 μm (diameter) by varying the flow rate of the dispersed phase. Mitoxantrone was used as the model drug in the drug release study. The encapsulation rate of the drug was 71% for chitosan particles, and 69% for magnetic iron oxide-chitosan particles, respectively. The iron oxide-chitosan composite particles had a faster release rate (up to 41.6% at the third hour) than the chitosan particles (about 24.6%). These iron oxide-chitosan composite particles are potentially useful for biomedical applications, such as magnetic responsive drug carriers, magnetic resonance imaging (MRI) enhancers, in the future.

The bi-functional nuclease AtBFN2 from Arabidopsis thaliana (EC 3.1.30.1) depends on zinc ion for cleaving single stranded DNA and RNA to yield 5′-nucleotides. It is a glycoprotein that participates in plant development and differentiation. The crystal structure of AtBFN2 shows a bound sulfate ion in the active site, at the center of the tri-nuclear cluster of zinc ions. The protein folds into a mostly α-helical structure with five short β-strands and contains four disulfide bonds. The zinc ions are coordinated to the side chains of three Asp and five His residues, two backbone atoms of Trp1, the sulfate ion, and a water molecule. An adenine base is bound adjacent to the active site and stacks with Tyr59. The core sugar residues attached to the three N-glycosylation sites of Asn91, Asn110 and Asn184 are also observed. By comparison with the nuclease P1 structure (PDB ID: 1AK0), the AtBFN2-sulfate-adenine complex model suggests a similar catalytic mechanism, in which the reaction starts with in-line attack at the phosphate by a zinc-activated water molecule.

Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3-5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50-60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

Rice starch can be hydrolyzed into maltose for trehalose bioconversion by enzymatic methods. In this study, we have successfully established an efficient production system for our recombinant PTTS in large scale. Three bio-treatments were developed to simplify the separation and purification of trehalose from complex rice saccharified liquid. The trehalose conversion rate of 64.63±4.05% at 30 °C can be reached using rice hydrolysate as the substrate in a 5l fermentor system. By 1% of raw material koji fermentation, the highest concentration of bioethanol (3.61±0.07%) was obtained at 30 °C for 36 h. After 12h of reaction time, the gluconic acid (24.47±0.33 mM) was successfully produced by glucose oxidase (40 U/g rice) using residual glucose as a substrate. After the batch/continuous ionic exchange process, the trehalose can be successfully separated, crystallized and identified as 92.6±0.02% purity and 94.2% of the recovery yield, respectively.

The oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) is an important fruit crop in the tropical and subtropical regions. However, oriental melon production is severely decreased by fungal diseases. In this study, antifungal protein (AFP) and chitinase (CHI) fusion genes were introduced into oriental melons to control fungal diseases caused by Rhizoctonia solani and Fusarium oxysporum. Transformation of oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) with Agrobacterium tumefaciens strain LBA4404 containing antifungal protein (AFP) and chitinase (CHI) fusion genes under the control of the cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene as a selectable marker was performed. Cotyledon explants of oriental melon were inoculated by Agrobacterium suspensions with pBI121–AFP–CHI and cultured in a regeneration medium. After regeneration, genomic DNA polymerase chain reaction (PCR) was conducted to confirm the presence of putative transgenic shoots. Southern blot analysis confirmed that the AFP–CHI fusion gene was incorporated into the genomic DNA of the PCR-positive lines. RT-PCR analysis showed that the AFP–CHI fusion gene was expressed in the individual transgenic lines. Western blot analysis revealed the accumulation of CHI protein in leaves. A segregation analysis of the T1 generation confirmed the inheritance of the transgene. Our results demonstrated that the AFP–CHI fusion gene was effective in protecting the transgenic melon plants against fungal disease caused by Rhizoctonia solani and Fusarium oxysporum.

The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 × His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.

Thioesterase I/protease I/lysophospholipase L 1 (EC 3.1.2.2) of Escherichia coli is an enzyme with multiple functions and is abbreviated as TAP due its unique discovery in at least 5 different laboratories since the 1960s. Crystal structural analysis identified essential amino acid residues for the catalytic triad, which are residues Ser10, Asp154 and His157, and the oxyanion hole, which contains residues Ser10, Gly44 and Asn73. Through site-directed mutagenesis, His157 was determined to play a pivotal role in enzyme catalysis through dual functions in the catalytic triad and oxyanion intermediate formation. The study of L109P revealed that its adjacent residue, Pro110, dominates the substrate preference for π -bond containing substrates. The non-active site residue Trp23 interweaves the protein framework and dynamic motions during catalysis through hydrogen bonding and aromatic–aromatic interactions. TAP may be a model enzyme with exquisite structural features that displays subtle characteristics for its substrate preferences.

The Tubby-like protein (TULP) is ubiquitous among multicellular organisms but has extremely diversified functions. Although functional analyses of certain mammalian and model plant TULPs are available and phylogenies separately based on animal or plant TULPs have been reconstructed, the origin, evolutionary history, and functional divergence of TULPs remain obscure. The existence of TULP in unicellular organisms is also not found yet. In this study, 145 TULP homologs were identified from four eukaryotic supergroups—Excavata, Chromalveolata, Archaeplastida (plants), and Opistokonta (fungi and animals). In addition to the tubby domain, many TULPs also contain different motifs (F-box, WD40 repeat, or SOCS box) in their N-terminus regions, leading to their groupings into three classes, in which TULPs with no N-terminal modifications (class II) locate at the basal positions on the phylogeny. Our comparison data reveals that phosphatidylinositol 4,5-bisphosphate (PIP2) binding sites are conserved in class II TULPs, especially those that are animal derived, but not in TULPs from classes I and III. Differentiation at the three PIP2 binding sites implies that the function of class II TULPs differs from those of other two classes. We further demonstrate that only class II TULPs can be targeted to the plasma membrane, suggesting functional divergence and differentiation of TULPs. Functional divergence and differentiation of TULPs should have occurred after duplications and structural modifications around 1100 MYA as eukaryotic organisms started diversifying.

In this study an integrated process for the concomitant purification and immobilization of recombinant Picrophilus torridus trehalose synthase for the synthesis of trehalose was reported. A highly porous crosslinked polystyrene divinylbezene-based metal chelator was used for the purification and immobilization of the poly(His)-tagged enzyme. While the Cu(II)-loaded adsorbent showed the highest protein adsorption capacity, 5.39 ± 0.03 mg protein/g, the Co(II)-loaded adsorbent exhibited the highest specificity for the adsorption of the poly(His)-tagged trehalose synthase, giving a specific activity of 3.98 ± 0.50 U/mg protein, more than fourfold higher than that of the Cu(II)-loaded adsorbent. No significant differences in optimal operation pH, temperature, and thermostability were observed upon immobilization. Under optimal reaction conditions, a trehalose conversion yield of 64.1% was obtained within 6 h. A residual activity of approximately 80% was maintained after 24 cycles in a repeated-batch operation. The decline in residual activity was primarily due to the desorption of the recombinant trehalose synthase. The proposed integrated approach for enzyme purification and immobilization with the metal chelator is promising for the development of industrial processes for trehalose production.

An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 °C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 μM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) μM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.

Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.

Bacillus subtilis is most commonly employed for secretion of recombinant proteins. To circumvent the problems caused by using plasmids, the T7 expression system known for its high efficiency was rebuilt in B. subtilis. Accordingly, a markerless and replicon-free method was developed for genomic insertion of DNAs. By the act of homologous recombination via the guide DNA, a suicidal vector carrying the gene of interest was integrated into genomic loci of bacteria. Removal of the inserted selection marker and replicon flanked by FRT sites was mediated by the FLP recombinase. By using the mentioned system, B. subtilis strain PT5 was constructed to harbor a genomic copy of the spac promoter-regulated T7 gene 1 located at wprA (encoding the cell wall-associated protease). Similarly, the T7 promoter-driven nattokinase or endoglucanase E1 of Thermomonospora fusca genes were also integrated into mpr (encoding an extracellular protease) of strain PT5. Consequently, the integrant PT5/Mmp-T7N or PT5/MT1-E1 resulted in a "clean" producer strain deprived of six proteases. After 24 h, the strain receiving induction was able to secret nattokinase and endoglucanase E1 with the volumetric activity reaching 10860 CU/mL and 8.4 U/mL, respectively. This result clearly indicates the great promise of the proposed approach for high secretion of recombinant proteins in B. subtilis.

A new recombinant Picrophilus torridus TSase (PTTS) has the catalytic ability for the conversion of maltose to trehalose by intramolecular transglucosylation. For industrial applications, the high thermostability of the enzyme would be the most important property for reducing the microbial contamination and lower the production cost. Therefore, in this study, we substituted ten selected proline residues of PTTS which differ from two well-known thermostable TSases. Interestingly, we found that the N503 mutant type, namely N503P-PTTS, showed about 39% higher relative activity than that of the wild type at 65 °C for 120 min. The trehalose yield of mutant N503P-PTTS was 1.3-fold higher than that of the wild type with sweet potato starch as substrate at 50 °C for 4 h. This suggests that the proline site substitution technology used in this study is useful for altering enzyme properties and catalytic efficiency for possible industrial applications.

To establish a mass micropropagation procedure for Cephalotus follicularis, the effects of varying the strengths of solid Murashige and Skoog (MS) medium were investigated using subcultured shoot explants. After a 60-day primary culture from root mass, the regenerated shoot explants were subcultured every 60 days in solid MS medium. To facilitate shoot proliferation, liquid MS medium was applied with or without exogenous auxin and cytokinin. Our results demonstrate that shoot proliferation and survival of C. follicularis is most effective in modified MS (MMS) medium containing one-fifth or one-tenth strength macronutrients and full-strength micronutrients. Successful shoot proliferation and development of C. follicularis explants were obtained in one-fifth or one-tenth modified liquid MS medium without auxin and cytokinin or with addition of 5 μM indole 3-acetic acid/1 μM N6-benzyladenine for 45 days. The liquid medium consistently produced more explants than the solid medium and shortened the culturing time. Plantlets cultured in hormone-free one-fifth MMS medium developed greater root systems. Using the liquid culture we established, vigorous plants with extensive roots were obtained within 4 months. Plant survival in the greenhouse reached 100%.

Escherichia coli possesses a versatile protein with the enzyme activities of thioesterase I, protease I, and lysophospholipase L(1). The protein is dubbed as TAP according to the chronological order of gene discovery (TesA/ApeA/PldC). Our previous studies showed that TAP comprises the catalytic triad Ser(10), Asp(154), and His(157) as a charge relay system, as well as Gly(44) and Asn(73) residues devoted to oxyanion hole stabilization. Geometrically, about 10 A away from the enzyme catalytic cleft, Trp(23) showed a stronger resonance shift than the backbone amide resonance observed in the nuclear magnetic resonance (NMR) analyses. In the present work, we conducted site-directed mutagenesis to change Trp into alanine (Ala), phenylalanine (Phe), or tyrosine (Tyr) to unveil the role of the Trp(23) indole ring. Biochemical analyses of the mutant enzymes in combination with TAP's three-dimensional structures suggest that by interlinking the residues participating in this catalytic machinery, Trp(23) could effectively influence substrate binding and the following turnover number. Moreover, it may serve as a contributor to both H-bond and aromatic-aromatic interaction in maintaining the cross-link within the interweaving framework of protein.