• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.829-835
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• 2000

The ptsH and ptsI genes of Lactococus lactis subsp. lactis ATCC 7962 (L. lactis 7962), encoding the general proteins of phosphotransferase system (PTS) components, HPr and enzyme I, respectively, were cloned and characterized. A 1.3 kb PCR product was obtained using a primer set that was hybridized to the internal region of the L. lactis 7962 pts HI genes and then subcloned into a low-copy number vector, pACYC184. The 5' upstream and 3' downstream region from the 1.3 kb fragment were subsequently clone using the chromosome walking method. The complete ptsHI operon was constructed and the nucleotide sequences determined. Two ORFs corresponding to HPr (88 amino acids) and enzyme I (575 amino acids) were located. The ptsHI genes of L. lactis 7962 showed a very high homology (84-90%) with those genes from other Gram-positive bacteria. A primer extension analysis showed that the transcription started at either one of two adjacent bases upstream of the start codon. Using a Northern analysis, two transcripts were detected; the first, a 0.3 kb transcript corresponding to ptsH and the second, a 2 kb transcript corresponding to ptsH and ptsI. The transcription level of ptsH was higher than that of ptsI. The concentration of the ptsH transcript in cells grown on glucose was similar to that in cells grown on lactose, yet higher than that in cells grown on galactose. The ptsI transcript was scarcely detected in cell grown on lactose or galactose. The ptsI transcript was scarcely detected in cells grown on lactose or galactose. The results of a sequence analysis and Northern blot confirmed that the ptsH and ptsI genes of L. lactis 7962 were arranged in an operon like other known ptsHI genes and the expression of the ptsHI genes was regulated at the transcriptional level in response to the carbon source.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1720-1728
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• 2020

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40℃, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.5
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• pp.614-628
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• 2019

Objective: The inhibitory leukocyte immunoglobulin-like receptors (LILRBs) play an important role in innate immunity. The present study represents the first description of the cloning and structural and functional analysis of LILRB1 and LILRB3 isolated from two genetically disparate chicken lines. Methods: Chicken LILRB1-3 genes were identified by bioinformatics approach. Expression studies were performed by transfection, quantitative polymerase chain reaction. Signal transduction was analyzed by western blots, immunoprecipitation and flow cytometric. Cytokine levels were determined by enzyme-linked immunosorbent assay. Results: Amino acid homology and phylogenetic analyses showed that the homologies of LILRB1 and LILRB3 in the chicken line 6.3 to those proteins in the chicken line 7.2 ranged between 97%-99%, while homologies between chicken and mammal proteins ranged between 13%-19%, and 13%-69%, respectively. Our findings indicate that LILRB1 and LILRB3 subdivided into two groups based on the immunoreceptor tyrosine-based inhibitory motifs (ITIM) present in the transmembrane domain. Chicken line 6.3 has two ITIM motifs of the sequence LxYxxL and SxYxxV while line 7.2 has two ITIM motifs of the sequences LxYxxL and LxYxxV. These motifs bind to SHP-2 (protein tyrosine phosphatase, non-receptor type 11) that plays a regulatory role in immune functions. Moreover, our data indicate that LILRB1 and LILRB3 associated with and activated major histocompatibility complex (MHC) class I and ${\beta}2-microglobulin$ and induced the expression of transporters associated with antigen processing, which are essential for MHC class I antigen presentation. This suggests that LILRB1 and LILRB3 are transcriptional regulators, modulating the expression of components in the MHC class I pathway and thereby regulating immune responses. Furthermore, LILRB1 and LILRB3 activated Janus kinase2/tyrosine kinase 2 (JAK2/TYK2); signal transducer and activator of transcription1/3 (STAT1/3), and suppressor of cytokine signaling 1 genes expressed in Macrophage (HD11) cells, which induced Th1, Th2, and Th17 cytokines. Conclusion: These data indicate that LILRB1 and LILRB3 are innate immune receptors associated with SHP-2, MHC class I, ${\beta}2-microglobulin$, and they activate the Janus kinase/signal transducer and activator of transcription signaling pathway. Thus, our study provides novel insights into the regulation of immunity and immunopathology.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2087-2097
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• 2016

Most cold-adapted enzymes possess higher $K_m$ and $k_{cat}$ values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near $K_m$. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately $30^{\circ}C$ and $40^{\circ}C$, respectively. The melting points were $33^{\circ}C$ and $45^{\circ}C$ for CpSK and EcSK, respectively. The ${\Delta}G_{H_2O}$ (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower $K_m$ for its substrate shikimate than most mesophilic SKs, and the $k_{cat}$ was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1650-1655
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• 2009

The $\beta$-glucuronidase (GUS) gene from Lactobacillus brevis RO1 was cloned and expressed in Escherichia coli GMS407. The GUS gene was composed of 1,812 bp, encoding a 603-amino-acid protein belonging to glycosyl hydrolase family 2 with three conserved domains. The amino acid similarity was higher than 70% with the $\beta$-glucuronidases of various microorganisms, yet less than 58% with the $\beta$-glucuronidase of L. gasseri ADH. Overexpression and purification of the GUS was performed in $\beta$-glucuronidase-deficient E. coli GMS407. The purified GUS protein was 71 kDa and showed 1,284 U/mg of specific activity at optimum conditions of pH 5.0 and $37^{\circ}C$. At $37^{\circ}C$, the GUS remained stable for 80 min at pH values ranging from 5.0 to 8.0. The purified enzyme exhibited a half-life of 1 h at $60^{\circ}C$ and more than 2 h at $50^{\circ}C$. When the purified GUS was applied to transform baicalin and wogonoside into their corresponding aglycones, $150\;{\mu}M$ of baicalin and $125\;{\mu}M$ of wogonoside were completely transformed into baicalein and wogonin, respectively, within 3 h.

• BMB Reports
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• v.40 no.2
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• pp.247-260
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• 2007

Cinnamate 4-hydroxylase (C4H) is a key enzyme of phenylpropanoid pathway, which synthesizes numerous secondary metabolites to participate in development and adaption. Two C4H isoforms, the 2192-bp BnC4H-1 and 2108-bp BnC4H-2, were cloned from oilseed rape (Brassica napus). They both have two introns and a 1518-bp open reading frame encoding a 505-amino-acid polypeptide. BnC4H-1 is 57.73 kDa with an isoelectric point of 9.11, while 57.75 kDa and 9.13 for BnC4H-2. They share only 80.6% identities on nucleotide level but 96.6% identities and 98.4% positives on protein level. Showing highest homologies to Arabidopsis thaliana C4H, they possess a conserved p450 domain and all P450-featured motifs, and are identical to typical C4Hs at substrate-recognition sites and active site residues. They are most probably associated with endoplasmic reticulum by one or both of the N- and C-terminal transmembrane helices. Phosphorylation may be a necessary post-translational modification. Their secondary structures are dominated by alpha helices and random coils. Most helices locate in the central region, while extended strands mainly distribute before and after this region. Southern blot indicated about 9 or more C4H paralogs in B. napus. In hypocotyl, cotyledon, stem, flower, bud, young- and middle-stage seed, they are co-dominantly expressed. In root and old seed, BnC4H-2 is dominant over BnC4H-1, with a reverse trend in leaf and pericarp. Paralogous C4H numbers in Brassicaceae genomes and possible roles of conserved motifs in 5' UTR and the 2nd intron are discussed.

• Journal of Microbiology and Biotechnology
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• v.19 no.8
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• pp.823-828
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• 2009

An endo-${\beta}-1$,4-xylanase (${\beta}$-xylanase) from Trichoderma harzianum C4 was purified without cellulase activity by sequential chromatographies. The specific activity of the purified enzyme preparation was 430 units/mg protein on D-xylan. The complementary DNA (cDNA) encoding ${\beta}$-xylanase (xynII) was amplified by PCR and isolated from cDNA PCR libraries constructed from T. harzianum C4. The nucleotide sequence of the cDNA fragment contained an open reading frame of 663 bp that encodes 221 amino acids, of which the mature protein is homologous to several ${\beta}$-xylanases II. An intron of 63 bp was identified in the genomic DNA sequence of xynII. This gene was expressed in Saccharomyces cerevisiae strains under the control of adh1 (alcohol dehydrogenase I) and pgk1 (phosphoglycerate kinase I) promoters in 2 ${\mu}$-based plasmids, which could render recombinants able to secrete ${\beta}$-xylanase into the media.

• Food Science and Biotechnology
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• v.18 no.3
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• pp.776-781
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• 2009

A putative cyclomaltodextrinase (BHCD) gene was found from the genome of Bacillus halodurans C-125, which encodes 578 amino acids with a predicted molecular mass of 67,279 Da. It shares 42-59% of amino acid sequence identity with common cyclomaltodextrinase (CDase)-family enzymes. The corresponding gene was cloned by polymerase chain reaction (PCR) and the dimeric enzyme with C-terminal 6-histidines was successfully overproduced and purified from recombinant Escherichia coli. BHCD showed the highest activity against ${\beta}-CD$ at pH 7.0 and $50^{\circ}C$. Due to its versatile hydrolysis and transglycosylation activities, BHCD has been confirmed as a member of CDases. However, BHCD can be distinguished from other typical CDases on the basis of its novel multisubstrate specificity. While typical CDases have over 10 times higher activity on ${\beta}-CD$ than starch or pullulan, the CD-hydrolyzing activity of BHCD is only 2.3 times higher than pullulan. In particular, it showed significantly higher activity ratio of maltotriose to acarbose than other common CDase-family enzymes.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1653-1663
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• 2010

The first gene (${\alpha}$-gal1) encoding an extracellular ${\alpha}$-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The ${\alpha}$-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal ${\alpha}$-galactosidases belonging to glycosyl hydrolase family 27. The ${\alpha}$-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant ${\alpha}$-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at $70^{\circ}C$, pH 4.5, and lost no activity over 10 days at $50^{\circ}C$. ${\alpha}$-Gal1 followed Michaelis-Menten kinetics ($V_{max}\;of\;240.3{\mu}M/min/mg,\;K_m\;of\;0.294 mM$) and was inhibited competitively by galactose ($K_m{^{obs}}$ of 0.57 mM, $K_i$ of 2.77 mM). The recombinant T. emersonii ${\alpha}$-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the ${\alpha}$-galactosidic linkage. Owing to its substrate preference and noteworthy stability, ${\alpha}$-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.