An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase.

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers.

Phenylalanine ammonia-lyase (PAL) catalyzes the nonoxidative deamination of phenylalanine to yield trans-cinnamic acid and ammonia. Recombinant Bambusa oldhamii BoPAL1/2 proteins were immobilized onto electrospun nanofibers by dextran polyaldehyde as a cross-linking agent. A central composite design (CCD)-response surface methodology (RSM) was utilized to optimize the electrospinning parameters. Escherichia coli expressed eBoPAL2 exhibited the highest catalytic efficiency among four enzymes. The optimum conditions for fabricating nanofibers were determined as follows: flow rate of 0.10 mL/h, voltage of 13.8 kV, and distance of 13 cm. The response surface models were used to obtain the smaller the fiber diameters as well as the highest PAL activity in the enzyme immobilization. Compared with free BoPALs, immobilized BoPALs can be reused for at least 6 consecutive cycles. The remained activity of the immobilized BoPAL proteins after storage at 4 °C for 30 days were between 75 and 83%. In addition, the tolerance against denaturants of the immobilized BoPAL proteins were significantly enhanced. As a result, the dextran polyaldehyde natural cross-linking agent can effectively replace traditional chemical cross-linking agents for the immobilization of the BoPAL enzymes. The PAL/nylon 6/polyvinyl alcohol (PVA)/chitosan (CS) nanofibers made are extremely stable and are practical for industrial applications in the future.

Cloning and characterization of the Bambusa oldhamii BoMDH-encoded malate dehydrogenase.

Malate dehydrogenase (MDH), which is ubiquitously occurred in nature, catalyzes the interconversion of malate and oxaloacetate. Higher plants contain multiple forms of MDH that differ in coenzyme specificity, subcellular localization and physiological function. A putative Bambusa oldhamii BoMDH cDNA was screened with the specific probe from the bamboo cDNA library. Sequence alignment shows that there's a high homology between the deduced amino acid sequence of BoMDH and MDH protein in Oryza sativa glyoxysome (92%). A 57 kDa fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the recombinant protein. The molecular mass of recombinant BoMDH was estimated to be 120 kDa, and the subunit form was 57 kDa by denatured SDS-PAGE, indicating that BoMDH presents as a homodimer. The optimum temperature and pH for BoMDH activity were 40 °C and 9.5, respectively. The Km values of BoMDH for malate and NAD+ were 5.2 mM and 0.52 mM. The kcat/Km values of BoMDH for malate and NAD+ were 163 min-1 mM-1 and 3060 min-1 mM-1.

Evaluation of aqueous ammonia pretreatment for enzymatic hydrolysis of different fractions of bamboo shoot and mature bamboo.

The production of fermentable sugars from different fractions of bamboo shoots and mature bamboos (Phyllostachys heterocycla var. pubescens) by cellulase and/or xylanase was investigated. Aqueous ammonia pretreatment exhibited high but different delignification capacities for different bamboo fractions. Supplementation of cellulases with xylanase synergistically improved the glucose and xylose yields of mature bamboo fractions. High hydrolyzability was observed in the hydrolysis of both non-pretreated and pretreated bamboo shoot fractions, suggesting pretreatment was not necessary for the hydrolysis of bamboo shoots. High hydrolyzability together with the advantages of low lignin content, fast growth, and widely distribution demonstrated that bamboo shoots were excellent lignocellulosic materials for the production of bioethanol and other biochemicals.

In vitro and in vivo safety evaluation of low molecular weight chitosans prepared by hydrolyzing crab shell chitosans with bamboo shoots chitosanase.

Our previous study demonstrated that the oral administration of low molecular weight chitosans (LMWC), prepared by hydrolyzing crab shell chitosans with bamboo shoots chitosanase in an appropriate dose, reduced aristolochic acid-induced renal lesions in mice. The objectives of this study were to evaluate the safety of LMWC using genetic and animal toxicity assays. Two assays for genotoxicity were performed: the chromosomal aberration of Chinese hamster ovary cells (CHO-K1 cells) (in vitro) and micronucleus assays in mice (in vivo). Acute oral toxicity and 28-day repeated feeding toxicity tests were performed via the oral gavage method in Sprague-Dawley (SD) rats. LMWC did not induce an increase in micronucleus ratios in vivo, and the chromosome aberration assay indicated that the LMWC was safe in terms of clastogenicity in doses up to 5.0 mg/ml. No acute lethal effect at a maximum tested dose of 5.0 g LMWC/kg body weight (bw) was observed in rats. The results of the 28-day study revealed no adverse effects on the body weight, feed consumption, hematology, blood biochemical parameters, organ weights or pathology. The no observed adverse effect level (NOAEL) of LMWC in rats was 1.0 g/kg bw for the subacute toxicity study.

Functional characterization of a serine-threonine protein kinase from Bambusa balcooa that implicates in cellulose overproduction and superior quality fiber formation.

BACKGROUND: Molecular markers allow rapid identification of biologically important germplasm/s having desired character. Previously we have reported a genotype specific molecular marker, Balco1128 [GenBank ID EU258678] of Bambusa balcooa containing an ORF (375 bp) having high similarity with receptor like cytoplasmic kinase of Arabidopsis and Oryza. Balco1128 was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. Under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development. RESULTS: The full length cDNA (3342 bp) of BbKst, a serine-threonine protein kinase was isolated from B. balcooa comprising of six LRR domains at the N-terminal end and a kinase domain at the C-terminal end. Bacteria-expressed BbKst-kinase domain (3339 bp long) showed Mg(2+) dependent kinase activity at pH 7.0, 28°C. Bioinformatics study followed by phospho-amino analysis further confirmed that BbKst-kinase belongs to the serine/threonine protein kinase family. Transcript analysis of the BbKst gene following RNA slot blot hybridization and qPCR revealed higher expression of BbKst during initiation and elongation stages of fiber development. Tissue specific expression studies showed much higher expression of BbKst transcript in stems and internodes of B. balcooa than in leaves and rhizomes. Southern analysis revealed single copy insertion of BbKst in most of the Agrobacterium mediated transgenic tobacco plants. Real-time PCR detected 150-200 fold enhanced expression of BbKst in different T1 tobacco lines than that of the vector transformed plants. Heterologous expression of BbKst under control of 35S promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. Number of xylary fibers was higher in transgenic T0 and T1 plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower Runkel ratio of the fibers. CONCLUSIONS: This finding clearly demonstrated that BbKst gene (GenBank ID JQ432560) encodes a serine/threonine protein kinase. BbKst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant.

Differential expression of genes encoding acid invertases in multiple shoots of bamboo in response to various phytohormones and environmental factors.

The promoter regions of two cell wall invertase genes, Boßfruct1 and Boßfruct2, and a vacuolar invertase gene, Boßfruct3, in Bambusa oldhamii were cloned, and putative regulatory cis-elements were identified. The expression of these three genes in multiple shoots of bamboo that were cultured in vitro under different conditions was analyzed by real-time PCR. The two cell wall invertase genes were upregulated by indole-3-acetic acid and cytokinins but responded differently to other phytohormones and different temperatures. Boßfruct1 was also upregulated by sucrose and glucose. In contrast, the Boßfruct2 expression was induced by the depletion of sucrose, and this induction could be suppressed by glucose and sucrose. The expression of Boßfruct3 was light-dependent; however, abscisic acid (ABA) could induce its expression in the dark. ABA and light exhibited an additive effect on the expression of Boßfruct3. Our results suggest that these three Boßfruct genes have individual roles in the adaption of the plant to environmental changes. Boßfruct2 might also have an essential role in the immediate response of cells to sucrose availability and in the maintenance of sink activity. Moreover, Boßfruct3 might be one of the interacting nodes of the light and ABA signaling pathways.

Effect of heat treatment on lignification of postharvest bamboo shoots (Phyllostachys praecox f. prevernalis.).

In order to evaluate the effects of heat treatment on the quality of postharvest bamboo shoots, the firmness, disease incidence, respiration and ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities, lignin and cellulose contents, and the activities of phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) were examined during storage at 20°C after heat treatment at 45°C for 5h. Heat treatment inhibited disease incidence and respiration, retarded ethylene production, and decreased ACS and ACO activities in bamboo shoots. Furthermore, heat treatment significantly delayed the rise in the activities of PAL, CAD and POD, which were associated with the inhibition of the synthesis of lignin and the delayed tissue lignification. These findings suggest that heat treatment could be a potential tool to delay lignification and decrease disease incidence in bamboo shoots during storage at 20°C.

Molecular cloning, expression pattern, and putative cis-acting elements of a 4-coumarate: CoA ligase gene in bamboo (Neosinocalamus affinis)

Background: 4-coumarate:CoA ligase (4CL) plays an important role at the divergence point from general phenylpropanoid metabolism to several branch pathways. Although 4CL sin higher plants have been extensively studied, little has known about the 4CL gene of bamboo. Results: In current study, a Na4CL gene putative encoding 4-coumarate:CoA ligase (4CL) and its 5’-flanking region were isolated from bamboo (Neosinocalamus affinis) by RACE-PCR and genomic DNA walker, respectively. Na4CL encodes a predicted protein of 557 amino acids, with conserved motifs of adenylate-forming enzymes. Phylogenetic analysis showed that Na4CL shared 62~85 percent identity with other known plant 4CLs, and cluster closely with some known 4CLs in monocots. Sequence analysis revealed conserved cis-acting elements (Box A and AC-II element) present in the Na4CL promoter. Additionally, a Na4CL RNAi construct was transformed into tobacco. Transgenic tobaccos displayed significant down-expression of endogenesis 4CL and reduced lignin contents. Conclusion: These results contribute to the knowledge of the presence of Na4CL gene and its possible role in phenylpropanoid metabolism.

Sucrose induces rapid activation of CfSAPK, a mitogen-activated protein kinase, in Cephalostachyum fuchsianum Gamble cells.

Sucrose was recently demonstrated to function as a molecular signal. However, sucrose-specific sensing and signalling pathways remain largely undefined. Here, we show that Cephalostachyum fuchsianum sucrose-activated protein kinase (CfSAPK) is transiently and specifically activated by sucrose in C. fuchsianum Gamble suspension cells. The result suggested that CfSAPK participates in a sucrose-signalling pathway. CfSAPK was partially purified from sucrose-treated cells and further analysed. Kinase activity assays revealed that CfSAPK preferentially used myelin basic protein (MBP) as substrate in vitro and strongly phosphorylate MBP threonine residue(s) and weakly phosphorylated MBP serine residue(s). Of the divalent cations tested, Mg(2+) was required for CfSAPK activation. Phosphatase treatment of CfSAPK abolished its kinase activity, indicating that phosphorylation is required for CfSAPK activation. Seven internal tryptic peptides identified from CfSAPK matched mitogen-activated protein kinases (MAPKs) in plants. CfSAPK cDNA was cloned using RT-PCR and rapid amplification of cDNA ends (RACE). CfSAPK cDNA encodes a 382-amino acid protein with a calculated molecular mass of 43,466.9 Da. The CfSAPK protein contains all 11 conserved kinase subdomains found in other Ser/Thr kinases. The amino acids sequence of CfSAPK is highly homologous to group A MAPKs in monocotyledon plants.

Purification and characterization of two chitosanase isoforms from the sheaths of bamboo shoots.

Two thermally stable chitosanase isoforms were purified from the sheaths of chitosan-treated bamboo shoots. Isoforms A and B had molecular masses of 24.5 and 16.4 kDa and isoelectric points of 4.30 and 9.22, respectively. Using chitosan as the substrate, both isoforms functioned optimally between pH 3 and 4, and the optimum temperatures for the activities of isoforms A and B were 70 and 60 °C, respectively. The kinetic parameters K(m) and V(max) for isoform A were 0.539 mg/mL and 0.262 µmol/min/mg, respectively, and for isoform B were 0.183 mg/mL and 0.092 µmol/min/mg, respectively. Chitosans were susceptible to degradation by both enzymes and could be converted to low molecular weight chitosans between 28.2 and 11.7 kDa. Furthermore, the most susceptible chitosan substrates were 50-70 and 40-80% deacetylated for isoforms A and B, respectively. Both enzymes could also degrade chitin substrates with lower efficacy. N-Bromosuccinimide and Woodward's reagent K strongly inhibited both enzymes.

Characterization of copper/zinc and manganese superoxide dismutase in green bamboo (Bambusa oldhamii): Cloning, expression and regulation.

Bamboo is distinguished by its rapid growth, for growth more than 100 cm per day. Because of the rapid growth, tissues have significant ATP requirements, which results in intense reduction of oxygen and thus oxidative stress. For this reason, bamboo may have a special and efficient scavenger system to release the stress during fast cell division and elongation. Here, we investigated superoxide dismutase (SOD, E.C.1.15.1.1), the first line of antioxidant enzymes, in green bamboo (Bambusa oldhamii). The SOD activity profile in this species was complex, with 5 genes and 7 isozymes of CuZnSOD and 4 genes and 1 isozyme of MnSOD. We isolated one of each of the green bamboo CuZnSOD and MnSOD genes, and their activities were stable under a broad range of pH and temperature treatments, even at room temperature for more than 3 days. Bamboo SODs showed developmental and tissue-specific regulation, and both transcript and protein levels were responsive to abscisic acid, UV-B and high-light treatments. The complexity of the cis-elements in promoter regions implied that the regulation mechanisms of SOD might help accomplish the unique fast-growth phenotype of green bamboo.

Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii.

Phenylalanine ammonia-lyase is the first enzyme of general phenylpropanoid pathway. A PAL gene, designated as BoPAL1, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL1 was 2,139 bp in size and predicted to encode a 712-amino acid polypeptide. BoPAL1 was the first intronless PAL gene found in angiosperm plant. Several putative cis-acting elements such as P box, GT-1motif, and SOLIPs involved in light responsiveness were found in the 5'-flanking sequence of BoPAL1 which was obtained by TAIL-PCR method. Recombinant BoPAL1 protein expressed in Pichia pastoris was active. The optimum temperature and pH for BoPAL1 activity was 50°C and 9.0, respectively. The molecular mass of recombinant BoPAL1 was estimated as 323 kDa using gel filtration chromatography and the molecular mass of full-length BoPAL was about 80 kDa, indicating that BoPAL1 presents as a homotetramer. The Km and kcat values of BoPAL1 for L-Phe were 1.01 mM and 10.11 s(-1), respectively. The recombinant protein had similar biochemical properties with PALs reported in other plants.

Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The K(m) of bamboo shell PAL for L-Phe was 476 µM, and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa, indicating that PAL from bamboo also exists as a tetramer. The optimum temperature for PAL activity was 50°C and the optimal pH 9.0. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Four PAL genes, designated as BoPAL1 to BoPAL4, were cloned from B. oldhamii. The open reading frames of BoPAL3 and BoPAL4 were 2142 and 2106 bp in size, respectively: BoPAL2-4 contained one intron and two exons, but no intron was found in BoPAL1. BoPAL4 expressed in Escherichia coli possessed both PAL and tyrosine ammonia-lyase activities. While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of BoPAL1 F133H and BoPAL2 F134H, respectively, showed decreased k(cat)/K(m) values toward L-Phe, whereas BoPAL2 F134H showed a slightly increased k(cat)/K(m) value toward L-Tyr. These data suggest other residues largely control Phe/Tyr substrate specificity. An antibody raised against the purified shell PAL was generated for histochemical studies. In bamboo shell and branch shoots, PAL was localized primarily in sclerenchyma cells.

Cloning and characterization of an antifungal class III chitinase from suspension-cultured bamboo ( Bambusa oldhamii ) cells.

A class III chitinase cDNA (BoChi3-1) was cloned using a cDNA library from suspension-cultured bamboo ( Bambusa oldhamii ) cells and then transformed into yeast ( Pichia pastoris X-33) for expression. Two recombinant chitinases with molecular masses of 28.3 and 35.7 kDa, respectively, were purified from the yeast's culture broth to electrophoretic homogeneity using sequential ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography, and Con A-Sepharose chromatography steps. N-Terminal sequencing and immunoblotting revealed that both recombinant chitinases were encoded by BoChi3-1, whereas SDS-PAGE and glycoprotein staining showed that the 35.7 kDa isoform (35.7 kDa BoCHI3-1) was glycosylated and the 28.3 kDa isoform (28.3 kDa BoCHI3-1) was not. For hydrolysis of ethylene glycol chitin (EGC), the optimal pH values were 3 and 4 for 35.7 and 28.3 kDa BoCHI3-1, respectively; the optimal temperatures were 80 and 70 degrees C, and the K(m) values were 1.35 and 0.65 mg/mL. The purified 35.7 kDa BoCHI3-1 hydrolyzed EGC more efficiently than the 28.3 kDa isoform, as compared with their specific activity and activation energy. Both recombinant BoCHI3-1 isoforms showed antifungal activity against Scolecobasidium longiphorum and displayed remarkable thermal (up to 70 degrees C) and storage (up to a year at 4 degrees C) stabilities.

Molecular cloning and functional identification of invertase isozymes from green bamboo Bambusa oldhamii.

Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.

Molecular characterization and expression of four cDNAs encoding sucrose synthase from green bamboo Bambusa oldhamii.

Bamboo is distinguished by its rapid growth. To investigate sucrose metabolism in this plant, we cloned the cDNAs encoding sucrose synthase (SuS) from Bambusa oldhamii and investigated their expression in growing shoots and leaves. Four cDNA clones, BoSus1, BoSus2, BoSus3 and BoSus4, were isolated by screening a cDNA library from etiolated bamboo shoots. Recombinant BoSuS proteins were produced in Escherichia coli and purified by immobilized metal affinity chromatography and ultrafiltration. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the abundance of the transcript of each gene. BoSus1 and BoSus3 may be duplicate or homeologous genes, the sequences of which show high identity. Similarly, BoSus2 shows high identity with BoSus4. Kinetic analysis showed that the two BoSuS isoforms of each type had similar michaelis constant (Km) values for sucrose, but different values for UDP. The four genes were expressed in various bamboo organs but were differentially regulated. The increase in the abundance of their mRNA paralleled the growth rate of the bamboo. The results suggest that, in bamboo, SuS is encoded by at least four genes, each with a specific role in providing substrates for the polysaccharide biosynthesis and/or energy production necessary to support the rapid growth of this species.