Processing of XynE (110-kDa) of Aeromonas caviae ME-1 to 72-kDa xylanase in Escherichia coli transformant

An extracellular 72-kDa xylanase from an Escherichia coli transformant that harbored pXED30 carrying xynE of Aeromonas caviae ME-1 was purified by extraction following SDS-PAGE to electrophoretic homogeneity. The analysis of N-terminal 10 amino acid residues (Gly-Ala-Arg-Ala-Gln-Ala-Ala-Ala-Asp-Val) revealed a 72-kDa product derived by the cleavage of Gly26-Gly27 at the N-terminal region of 110 kDa XynE. The 72-kDa xylanase from A. caviae ME-1 was found to have the same sequence as that of the N-terminal 10 amino acid residues. When OmpT-deficient E. coli mutants were used as hosts, the 72-kDa xylanase was detected in cell-free extracts, but not in the culture supernatant, suggesting that OmpT is not involved in the cleavage at the C-terminal region, but is involved in the secretion of 72-kDa xylanase to the culture medium.     

坎皮纳斯类芽孢杆菌木聚糖酶的纯化以及酶学性质

采用硫酸铵盐析、Sephadex G-50凝胶过滤、DEAE Sepharose Fast Flow离子交换层析对坎皮纳斯类芽孢杆菌（Paenibacillus campinasensis）xy-7发酵液进行分离纯化,获得纯化的木聚糖酶,纯化倍数为26.36,酶活回收率为5.13%。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果为单一条带,分子质量为24.5 ku。酶学性质研究结果表明,该酶的最适反应温度为60 ℃,最适pH值为8.0。K+、Fe2+对酶有激活作用,Zn2+、Cu2+对酶有强烈的抑制作用。以榉木木聚糖为底物时,米氏常数Km=4.733 mg/mL,最大酶反应速率Vm=315.85 μmol/（min·mg）。     

Cloning of a thermostable xylanase from Actinomadura sp. S14 and its expression in Escherichia coli and Pichia pastoris

A thermophilic xylan-degrading Actinomadura sp. S14 was isolated from compost in Thailand. Hemicellulase activities such as endo-1,4-β-xylanase, β-xylosidase and α-arabinofuranosidase were induced with xylan-containing agriculture wastes and oat spelt xylan. The gene encoding xylanase consisting of 687bp was cloned from Actinomadura sp. S14. The deduced amino acid sequence contained a signal peptide of 41 amino acids and a probable mature xylanase of 188 amino acids. An open reading frame (xynS14) corresponding to a mature xylanase was expressed in Escherichia coli and Pichia pastoris. The specific activity of purified XynS14 (P. pastoris) was 2.4-fold higher than XynS14 (E. coli). Both XynS14s showed the same basic properties such as optimal pH and temperature (pH 6.0 and 80°C) and stability in a broad pH range (pH 5.0-11.0) and at high temperatures up to 80°C. Both XynS14s showed approximately the same substrate specificity and K(m) values toward various xylans, but XynS14 (P. pastoris) showed higher V(max) and K(cat) than XynS14 (E. coli). Higher specific activities of XynS14 (P. pastoris) may be due to protein-folding in the host. Purified XynS14 showed more endo-1,4-β-xylanase activity on xylan and xylooligosaccharides than on xylotriose.     

Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-beta-D-xyloside

Thermotoga maritima MSB8 possesses two xylanase genes, xynA and xynB. The xynB gene was isolated from the genomic DNA of T. maritima, cloned, and expressed in Escherichia coli. XynB was purified to homogeneity by heat treatment, affinity chromatography and ion-exchange column chromatography. The purified enzyme produced a single band upon SDS-PAGE corresponding to a molecular mass of 42 kDa. At 70 degrees C, the enzyme was stable between pH 5.0 and pH 11.4, and it was stable at temperatures of up to 100 degrees C from pH 7.0 to pH 8.5. At 50 degrees C, XynB displayed an optimum pH of 6.14 and at this pH the temperature for optimal enzyme activity was 90 degrees C. XynB exhibited broad substrate specificity and was highly active towards p-nitrophenyl-beta-D-xylobioside with K(m) and k(cat) values of 0.0077 mM and 5.5 s(-1), respectively, at 30 degrees C. It was also active towards p-nitrophenyl-beta-D-xyloside. The initial product of the cleavage of p-nitrophenyl-beta-D-xyloside was xylobiose, indicating that the major reaction in the cleavage was transglycosylation, not hydrolysis.     

Purification, characterization and amino acid sequencing of divergicin M35: a novel class IIa bacteriocin produced by Carnobacterium divergens M35

Carnobacterium divergens M35, isolated from a commercial sample of frozen smoked mussels, produces a new bacteriocin, divergicin M35, a class IIa bacteriocin. Divergicin M35 is sensitive to pronase-E, alpha-chymotrypsin and proteinase K, but not to trypsin and withstands thermal treatments up to 121 degrees C for 30 min. Divergicin M35 was extracted from the culture supernatant of C. divergens M35 using an SP-Sepharose cation-exchange column, desalted and purified on a C18 Sep-Pack column and further purified by reverse phase-high pressure liquid chromatography. This procedure allowed the recovery of 10% of the bacteriocin present in the culture supernatant with purity higher than 99%. Divergicin M35 had a molecular mass of 4518.75 Da as determined by mass spectrometry, a pI value of 8.3 and positive net charge (+3). The amino acid sequence of divergicin M35 was found to consist of 43 amino acid with four cysteine residues (Cys10, 15, 25, 43) and showed 80.5% homology with divercin V41 (80.5%) and 80.0% with bavaricin MN. Divergicin M35 showed powerful antilisterial activity, especially against Listeria monocytogenes and was also active against carnobacteria but not against strains of Lactococcus, Lactobacillus, Enterococcus, Bifidobacteria and Escherichia. Divergicin M35 production began in late exponential phase and reached a maximum activity of 65,000 AU/ml in early stationary phase. Initial broth pH, Tween 80 and acetate did not affect C. divergens M35 growth or divergicin production. This bacteriocin may be a potential tool for inhibiting L. monocytogenes in seafood products that do not usually undergo an adequate heat treatment.     

Purification and some properties of a chitinase from Xanthomonas sp. strain AK

Chitinase B (ChiB) was purified from the culture supernatant of Xanthomonas sp. strain AK by Phenyl-Toyopearl 650M and DEAE-Toyopearl 650M column chromatographies. The purified enzyme preparation gave a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight of ChiB was estimated to be 48,000. The enzyme was optimally active at pH 6.0 and 60 degrees C. N-Terminal amino acid sequence analysis suggested that ChiB is a member of glycosyl hydrolase family 18 and that it is genetically different from ChiA recently reported (Sakka et al., J. Ferment. Bioeng., 86, 527-533, 1998). Immunological analysis suggested that ChiB was the major chitinase species in the culture supernatant of Xanthomonas sp. strain AK and that production of the enzyme was induced by the presence of chitin.     

Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

Thermostable and alkaline-tolerant cellulase-free xylanase produced by thermotolerant Streptomyces sp. Ab106

Cellulase-free xylanase was produced by Streptomyces sp. Ab106 using cane bagasse as the substrate at 55 degrees C. Its maximum activity was 13 IU without cellulase and mannanase activities. Its profiles were investigated. Its optimum temperature and pH were 60 degrees C and 6.0, respectively. More than 70% of its activity was remained at 60 degrees C at pH 9. This enzyme was quite stable and exhibited an active of more than 70% for 144 h at 60 degrees C, and of more than 80% for 144 h at 40 degrees C, pH 9. This thermo-tolerant and alkaline-tolerant xylanase can be used in the pulp bleaching process.     

Primary structure and polymorphism of 2S albumins from seeds of Andean lupin (Lupinus mutabilis Sweet)

 2S albumins were isolated from seeds of Andean lupin (Lupinus mutabilis Sweet) by buffer extraction, ammonium sulphate precipitation and ultrafiltration followed ion-exchange and reversed-phase HPLC. The 2S albumin preparation (LM2S) contained eight albumins. The complete amino acid sequences of small and large subunits of three major albumins (LM2S-4, -5 and -6) were determined by automated Edman degradation of S-pyridylethylated polypeptides and peptides obtained from them by enzymatic digestions. The small subunit of the dominant 2S albumin (LM2S-4) contains 39 amino acid residues and has a molecular mass of 4731 Da. The large subunit of LM2S-4 contains 74 amino acid residues (molecular mass=8708 Da). Two 2S albumin isoforms (LM2S-4 and -6) are due to the expression of two distinct genes; LM2S-6 isoform has eight amino acid replacements when its sequence is compared with the sequence of LM2S-4. The LM2S-5 isoform contains an identical small subunit to LM2S-4, and has in comparison with LM2S-4 two additional amino acid residues at the N-terminus of the large subunit. The amino acid sequences of 2S isoforms from L. mutabilis showed high homology (78–83% identity) with 2S albumins from different Old World Lupinus species. Received: 15 December 1998 / Revised version: 9 February 1999     

Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization

Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.     

Characterisation of a thermostable xylanase from Chaetomium sp. and its application in Chinese steamed bread

A novel thermophilic xylanase-producing fungus, Chaetomium sp. CQ31 produced 131 U ml⁻¹ of xylanase when grown on a medium containing corncob (3.5%, w/v) at 37 °C for 6 days. A low molecular xylanase was purified 6.5-fold to homogeneity with a recovery yield of 17.5%. Its molecular mass was estimated to be 25.1 kDa by SDS–PAGE. The xylanase had an optimum pH of 7.5, and its optimal temperature was 65 °C. Apparent K_m values of the xylanase for birchwood, beechwood and oat-spelt xylan were 1.3, 0.86 and 4.4 mg/ml, respectively. The influence of this xylanase on the quality of Chinese steamed bread (CSB) was further studied. Addition of xylanase in the range 2.5–5.0 ppm caused a 20–24.5% increase in specific volume over the control and remarkable decrease (8.9–24.2%) in firmness was also noticed. This is the first report on the purification, characterisation and application of a xylanase from Chaetomium sp. CQ31.     

Molecular structure and gene analysis of Ce3+ -induced methanol dehydrogenase of Bradyrhizobium sp. MAFF211645

The molecular structure and nucleotide sequence of Ce(3+)-induced methanol dehydrogenase (MDH) of Bradyrhizobium sp. MAFF211645 were investigated. The addition of 30 μM Ce(3+) to 1/10 nutrient broth containing 0.5% methanol remarkably increased MDH activity. Furthermore, La(3+) increased MDH activity, but other heavier rare earth and metal elements did not have the same effect. MDH increased by Ce(3+) was purified by sequential column chromatography, and the purified MDH migrated as a single band with an apparent molecular weight of 68 kDa on SDS-PAGE. The apparent molecular weight of native MDH was estimated to be 108,000 by gel chromatography. The MDH was comprised of two identical subunits. N-terminal 23-amino acid sequence, 1-NDELHKMAQNPKDWVMPAGDYAN-23, of the purified MDH exhibited 91.3% identity to that of the MDH large subunit-like protein encoded by mxaF' of Bradyrhizobium japonicum USDA110. Nucleotide sequencing of the MDH gene of strain MAFF211645 yielded a deduced amino acid sequence comprising 601 amino acid residues, an N-terminal signal peptide, and a mature MDH comprising 578 amino acid residues with a predicted molecular mass of 62,918 Da. Further analysis of the deduced amino acid sequence of mature MDH revealed that the functional amino acids in its active site, such as two adjacent Cys residues, and bacterial quinoprotein signatures 1 and 2 were conserved. These results indicate that Ce(3+)-induced MDH encoded by mxaF' may be involved in methanol metabolism in Bradyrhizobium sp. MAFF211645.     

短小芽孢杆菌固态发酵生产木聚糖酶的培养条件优化

采用单因素试验和正交试验的方法研究了短小芽孢杆菌固态发酵生产木聚糖酶的培养条件,确定了利用麸皮作为在主要基质进行固态发酵生产木聚糖酶的适宜条件：pH9、温度35℃、料液比1：2、接种量10％。在500mL三角瓶中固态培养72h左右,木聚糖酶的活力可达4915U／g干曲。     

Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii) and elkhorn sculpin (Alcichthys alcicornis): Isolation and characterization

Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii), TR-J, and elkhorn sculpin (Alcichthys alcicornis), TR-E, were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The molecular weights of TR-J and TR-E were estimated to be 24,000 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. TR-J and TR-E revealed optimum temperatures of 60 and 50 °C, respectively, and showed the same optimum pH (pH 8.0) for hydrolysis of N-p-tosyl-l-arginine methyl ester. TR-J and TR-E were unstable at above 50 and 40 °C, respectively, and were more stable at alkaline pH than at acidic pH. Thermal stabilities of TR-J and TR-E were highly calcium dependent. These purified trypsin enzymes were inhibited by serine protease inhibitors, such as TLCK and soybean trypsin inhibitor. The N-terminal amino acid sequences of TR-J and TR-E were also investigated. The N-terminal amino acid sequences of TR-J, IVGGYECKPYSQPHQVSLNS, and TR-E, IVGGYECTPHSQAHQVSLNS, were found, and these sequences showed highly homology to other fish trypsins.     

高产木聚糖酶嗜热子囊菌固体发酵条件与酶学性质

以本实验室从土壤中分离筛选到的一株高产木聚糖酶的嗜热子囊菌QS7-2-4为生产菌种,进行固体发酵产木聚糖酶的发酵条件研究,结果表明最佳产酶条件为:玉米芯:麸皮为7:3(w/w);最佳氮源为酵母膏和胰蛋白胨的混合氮源,添加量为1.5%;吐温-80添加量为0.5%,初始pH为7.2,培养基含水量为80%,250mL三角瓶装料量为8g,发酵温度50℃,发酵时间72h,该条件下木聚糖酶产量达27952U/g干基。该酶最适反应温度为75℃,最适反应pH为4.5,在70℃以下具有良好的稳定性,在室温下储藏150d仍然保留87%的活性。     

Acidophilic xylanase from Aureobasidium pullulans: efficient expression and secretion in Pichia pastoris and mutational analysis

A yeast-like fungus Aureobasidium pullulans var. melanigenum strain ATCC 20524 produces an extracellular acidophilic endo-1,4-beta-xylanase with an optimum pH of 2.0 [Ohta et al., J. Biosci. Bioeng., 92, 262-270 (2001)]. The xynI cDNA encoding the precursor protein (XynI) was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase I gene promoter. The 34 amino acid prepro-signal peptide of the A. pullulans XynI directed the efficient secretion of 178 mg of active xylanase per liter of the culture medium. The secretion level of the xylanase with its own signal peptide was comparable to that of the mature protein fused to the prepro leader from Saccharomyces cerevisiae alpha-mating factor and twofold higher than that of the mature protein fused to the pre-type signal peptide from P. pastoris acid phosphatase. The N-terminal amino acid sequence and the apparent M(r) of 24 kDa of the secreted recombinant protein indicated the native-like processing of the A. pullulans XynI signal sequence in P. pastoris. The three-dimensional model and mutational analysis of the xynI gene product showed that Asp-73 and Glu-157 residues located at the upper and lower edges of the active site cleft, respectively, play a significant role in its low pH optimum.     

Purification and characterization of a 49-kDa chitinase from Streptomyces griseus HUT 6037

A 49-kDa chitinase (pI7.3) was purified to homogeneity from the culture supernatant of Streptomyces griseus HUT 6037 by ultrafiltration, DEAE-Sephadex A-50 and Sephadex G-100 column chromatographies, and chromatofocusing. The purified enzyme was stable up to 40 degrees C. The N-terminal amino acid sequence of the enzyme was highly homologous to the N-terminal region of the fibronectin type III-like domain of S. olivaceoviridis chitinase 01 belonging to family 18 glycosyl hydrolases. The 49-kDa chitinase hydrolyzed partially N-acetylated chitosan more easily than colloidal chitin. The hydrolyzate of 54% deacetylated chitosan by the enzyme was separated by CM-Sephadex C-25 column chromatography. The structures of the oligosaccharides obtained were determined by MALDI-TOF MS analysis combined with exo-glycosidase digestion. In addition to GlcNAc, (GlcNAc)2, and (GlcNAc)3, hetero-chitooligosaccharides with GlcNAc at the reducing end were detected. Thus, the specificity of the enzyme for the hydrolysis of the beta-1,4-glycosidic linkages in partially N-acetylated chitosan was similar to that of the family 18 chitinases.     

Purification, characterization, and gene cloning of sphingomyelinase C from Streptomyces griseocarneus NBRC13471

Sphingomyelinase C (SMC) was purified to homogeneity from the culture supernatant of Streptomyces griseocarneus NBRC13471. The purified enzyme appeared as a single band of 38 kDa by using an electropherogram trace. The molecular mass of the enzyme as determined by MALDI-TOF MS was 32,102 Da, indicating that SMC is monomeric in nature. Under experimental conditions, the highest enzyme activity was found at pH 9.0 and 50–55 °C, and the enzyme was stable from pH 5 to 10 and up to 37 °C. The SMC activity requires Mg²⁺ or Mn²⁺ and the order of potency to enhance the activity was Zn²⁺ ≥ Mn²⁺ > Cu²⁺ ≥ Fe²⁺. Phenylmethylsulfonyl fluoride and EDTA inhibited the enzyme activity, showing that SMC belongs to a group of metalloenzymes and a class of serine hydrolases. The enzyme activity was inhibited by DTT, but not by mercaptoethanol and iodoacetamide. SDS inhibited the enzyme activity; by contrast, Triton X-100 stimulated the activity. The N-terminal and internal amino-acid sequences were determined as H₂N-APAAATPSLK, AREIAAAGFFQGND, and NTVVQETSAP. The gene encoding SMC consisted of 1020 bp encoding a signal peptide of 42 amino acids and a mature protein of 297 amino acids with a calculated molecular mass of 32,125 Da. The conserved region of DNase I-like family enzymes and the amino acid residues that are highly conserved in the active center of other bacterial SMCs were also found in the deduced amino acid sequence of S. griseocarneus SMC.