Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

Summary The couplings ofN-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence ofBacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Amino acid modified chitosan beads: Improved polymer supports for immobilization of lipase from Candida rugosa

Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (k_cat/K_m) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads.     

Catalytic properties of the immobilized Talaromyces thermophilus β-xylosidase and its use for xylose and xylooligosaccharides production

The present study explores the efficiency of Talaromyces thermophilus β-xylosidase, in the production of xylose and xylooligosaccharides. The β-xylosidase was immobilized by different methods namely ionic binding, entrapment and covalent coupling and using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the best support material for β-xylosidase immobilization; it gave the highest immobilization and activity yields (94%, 87%, respectively) of initial activity, and also provided the highest stability, retaining 94% of its initial activity even after being recycled 25 times. Shifts in the optimal temperature and pH were observed for the immobilized β-xylosidase when compared to the free enzyme. The maximal activity obtained for the immobilized enzyme was achieved at pH 8.0 and 53 °C, whereas that for the free enzyme was obtained at pH 7.0 and 50 °C. The immobilized enzyme was more thermostable than the free β-xylosidase. We observed an increase of the K_m values of the free enzyme from 2.37 to 3.42 mM at the immobilized state. Native and immobilized β-xylosidase were found to be stimulated by Ca²⁺, Mn²⁺ and Co²⁺ and to be inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, EDTA and SDS. Immobilized enzyme was found to catalyze the reverse hydrolysis reaction, forming xylooligosaccharides in the presence of a high concentration of xylose. In order to examine the synergistic action of xylanase and β-xylosidase of T. thermophilus, these two enzymes were co-immobilized on chitosan. A continuous hydrolysis of 3% Oat spelt xylan at 50 °C was performed and better hydrolysis yields and higher amount of xylose was obtained.     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Performance of Aspergillus niger B 03 β-xylosidase immobilized on polyamide membrane support

The dynamics of β-xylosidase biosynthesis from Aspergillus niger B 03 was investigated in laboratory bioreactor. Maximum xylosidase activity 5.5 U/ml was achieved after 80 h fermentation at medium pH 4.0. The isolated β-xylosidase was immobilized on polyamide membrane support and the basic characteristics of the immobilized enzyme were determined. Maximum immobilization and activity yield obtained was 30.0 and 6.8%, respectively. A shift in temperature optimum and pH optimum was observed for immobilized β-xylosidase compared to the free enzyme. Immobilized enzyme exhibited maximum activity at 45 °C and pH 4.5 while its free counterpart at 70 °C and pH 3.5, respectively. Thermal stability at 40 and 50 °C and storage stability of immobilized β-xylosidase were investigated at pH 5.0. Kinetic parameters K_m, V_max and K_i were determined for both enzyme forms. Free and immobilized β-xylosidase were tested for xylose production from birchwood xylan. The substrate was preliminarily depolymerized with xylanase to xylooligosaccharides and the amount of xylose obtained after their hydrolysis with free and immobilized β-xylosidase was determined by HPLC analysis. Continuous enzyme hydrolysis of birchwood xylan was performed with xylanase and free or immobilized β-xylosidase. The maximum extent of hydrolysis was 25 and 30% with free and immobilized enzyme, respectively. Immobilized preparation was also examined for reusability in 20 consecutive cycles at 40 °C.     

Comparative Studies of the Indoleamine Dioxygenase-like Myoglobin from the Abalone Sulculus diversicolor

The abalone Sulculus diversicolor contains abundant myoglobin in its buccal mass. The myoglobin is homodimeric and the molecular mass of the constituent polypeptide chain is 41,000 Da. The amino acid sequence and gene structure are highly homologous with those of a vertebrate tryptophan-degrading enzyme, indoleamine dioxygenase (IDO). Thus Sulculus myoglobin evolved from an IDO gene, and represents a typical case of functional convergence. The oxygen equilibrium properties of Sulculus myoglobin were examined and compared with those of myoglobins from other sources. It binds oxygen reversibly, and the P₅₀ was determined to be 3.8 mmHg at 20°C and pH 7.4, showing that the oxygen affinity of Sulculus myoglobin is significantly lower than those of usual 16 kDa myoglobins. It also displays no cooperativity (n_max: 1.02–1.06) and no alkaline Bohr effect between pH 7.0 and 7.9. The cDNA-derived amino acid sequences of vertebrate IDOs, molluscan IDO-like myoglobins and a homolog in the yeast Saccharomyces were aligned, and several amino acid residues were proposed as candidates for key residues to control the function of IDO or myoglobin.     

拟南芥谷胱甘肽S-转移酶Zeta类进化酶的获得及其特性分析

拟南芥谷胱甘肽S-转移酶Zeta类(AtGSTZ)是一种与细胞代谢和环境净化密切相关的多功能酶.应用易错PCR和多轮DNA洗牌技术构建了AtGSTZ随机突变文库;再利用pH指示剂颜色改变法对突变文库进行筛选,获得了9个二氯乙酸脱氯活性提高的突变子.其中,NN23含25个氨基酸突变,比活力提高120%,NN20含24个氨基酸突变,比活力提高102%,EC1含2个氨基酸突变,比活力提高47%,其他6个为单点突变,比活力分别提高9%～60%.酶学分析显示,所有进化酶对底物二氯乙酸的催化效率和对谷胱甘肽的亲和力以及个别进化酶的复性能力都得到不同程度的提高,但热稳定性均没有明显改善.同时,对一系列与AtGSTZ空间折叠及催化活性相关位点进行了讨论.     

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA⁺)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.     

Occurrence of a unique amino acid racemase in a basidiomycetous mushroom, Lentinus edodes

Free -alanine was detected in a cell extract of the fruit-body of an edible basidiomycetous mushroom, Lentinus edodes (Shiitake), by means of reverse-phase high performance liquid chromatography. We also found an amino acid racemase activity in L. edodes fruit-body, and purified the enzyme. The enzyme has a molecular weight of approximately 86,000, and consists of two subunits of identical molecular weight (44,000). The optimal pH of the enzyme activity is around pH 9.5 for both -to- and -to- alanine racemization. The enzyme requires pyridoxal 5′-phosphate as a cofactor. K_m and V_max values for -alanine were 37.3 mM and 520 nmol/min/mg, respectively; for -alanine, they were 9.21 mM and 141 nmol/min/mg, respectively. The equilibrium constant was calculated to be 1.10, which is consistent with the theoretical value for the racemase reaction. The ability of the enzyme to catalyze the racemization of various -amino acids was investigated. The enzyme catalyzes the racemization of -serine (relative reaction rate, 144% of rate for -alanine), -alanine (100%), -homoserine (17.1%), -2-aminobutyrate (5.6%), -glutamate (4.5%), and -asparagine (3.2%). To the best of our knowledge, this is the first report of an amino acid racemase produced by a basidiomycetous mushroom.     

Peptide syntheses mediated by Bacillus subtilis protease

Bacillus subtilis protease (Amano protease N) was examined as a catalyst for peptide bond formation via both the kinetically and thermodynamically controlled approaches. In general, the latter approach proved to be superior to the former, and a series of dipeptide syntheses and several segment condensations were achieved in good to high yields using the immobilized enzyme on Celite in acetonitrile with low water content.     

Heterologous expression and characterization of man gene from Bacillus Subtilis in Pichia Pastoris

β-Mannanase catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannan, which are abundant in the cell wall structure of ungerminated leguminous seeds. The mature β-mannanase originated from Bacillus subtilis was expressed in Pichia pastoris, a methylotrophic yeast, using the leader peptide sequence of Saccharomyces cerevisiae α-factor. The cultivation of β-mannanase expressing Pichia pastoris yields up to 1.8 g/L protein. In the supernatant the activity of the 40 kDa—total mannanase attained a level of 1102.0 IU/mL. The properties of the β-mannanase were characterized. Optimum pH and temperature for the recombinant enzyme were 5.5 and 50°C respectively. The enzyme was stable at pH 5.0–10.0 and maintained over 30% original activity after incubating at 70°C for 30 min. __________ Translated from China Biotechnology, 2005, 26(7): 52–56 [译自: 中国生物工程杂志]     

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.     

Structure of the HOM2 gene of Saccharomyces cerevisiae and regulation of its expression

Summary In Saccharomyces cerevisiae the HOM2 gene encodes aspartic semi-aldehyde dehydrogenase (ASA DH). The synthesis of this enzyme had been shown to be derepressed by growth in the presence of high concentrations of methionine. In the present work we have cloned and sequenced the HOM2 gene and found that the promoter region of this gene bears one copy of the consensus sequence for general control of amino acid synthesis. This prompted us to study the regulation of the expression of the HOM2 gene. We have found that ASA DH is the first reported enzyme of the related threonine and methionine pathway to be regulated by the general control of amino acid synthesis.     

Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization

The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichia coli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Glyoxyl agarose can only immobilize enzymes via a multipoint and simultaneous attachment. Therefore, only proteins having several terminal amino groups in a position that permits their interaction with a flat surface can be immobilized. GDH became rapidly immobilized at pH 7 and its multimeric structure became stabilized as evidenced by SDS-PAGE. This derivative was stable at acidic pH value while the non-stabilized enzyme was very unstable under these conditions due to subunit dissociation. After immobilization, a further incubation at pH 10 improved enzyme stability under any inactivating conditions by increasing the enzyme–support bonds. In fact, GDH immobilized at pH 7 and incubated at pH 10 preserved more activity than GDH directly immobilized at pH 10 (50% versus 15% after 24 h of incubation) and was also more stable (1.5- to 3-fold, depending on the conditions).This method could be extended to any other multimeric enzyme expressed in mesophilic hosts.     

不吸水链霉菌葡萄糖异构酶的物化表征

葡萄糖异构酶是一种催化葡萄糖异构为果糖的酶。本文用紫外吸收光谱、红外光谱、氨基酸组分分析、聚丙烯酰胺凝胶梯度电泳、SDS-聚丙烯酰胺凝胶电泳、超薄 层聚丙烯酰胺凝胶等电聚焦电泳技术研究了不吸水链霉菌嗜热亚种M1033菌株产生的葡萄糖异构酶的一些物化性质。结果表明由本实验室制备的均一葡萄糖异构酶的A₂₈₀与A₂₆₀的比值是1.76。它是由一个亚单位组成的酶分子。最小分子量是49000。pI值是5.2。氨基酸组分与其它来源的葡萄糖异构酶的氨基酸组分相比较有一些差异,其中Glu,Gly,ALa和Leu的含量都此其它异构酶的高,而Met,Trp,Asp,Thr则比其它葡萄糖异构酶的低。     

Isolation and characteristics of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Phospholipase A₂ was purified from the pyloric ceca of the starfish Asterina pectinifera. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 20,000. The optimum pH and temperature of the enzyme were at around pH 9.0 and 50°C, respectively, and the activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca²⁺. The enzyme had no fatty acid specificity. Starfish phospholipase A₂ hydrolyzed phosphatidylcholine more effectively than phosphatidylethanolamine.     

Purification and characterization of a novel anti-fungal protein from Gastrodia elata

An anti-fungal protein GAFP-1 (Gastrodia anti-fungal protein, also called gastrodianin) was purified from Gastrodia elata B1. f. flavida S. Chow (Orchidaceae), a parasitic plant on the fungus Armillaria mellea. It can inhibit the hyphal growth of some phytopathogenic fungi such as Valsa ambiens, Rhizoctonia solani, Gibberella zeae, Ganoderma lucidum and Botrytis cinerea in vitro. GAFP-1 is a monomer with a molecular mass of 10 kDa and a pI of 8.45. The optimum pH for its inhibitory activity is 5.0 ∼ 6.0. GAFP-1 is insensitive to high temperatures. It can preserve 75% inhibitory activity after 30 min at 60°C. Amino acid composition analysis revealed that GAFP-1 is rich in Asp (22.1%), Gly (10.0 %) and Leu (9.4 %), and does not contain any Pro. The amino acid sequence of the N-terminal was determined and found to share high homology with those of other lectins from orchids such as Listera ovata and Epipactis helleborine. GAFP-1 could not agglutinate trypsin-treated rabbit erythrocytes. It could bind to chitin, immobilized mannose and N-acetylglucosamine in 50mM sodium acetate buffer (pH 5.0) with 2 M ammonium sulfate. These data suggest that GAFP-1 could be a lectin-like protein with strong inhibitory activity against certain fungal pathogens.     

Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus

The gene encoding the extracellular neutral metalloprotease ShpI from Staphylococcus hyicus subsp. hyicus was cloned. DNA sequencing revealed an ORF of 1317 nucleotides encoding a 438 amino acid protein with Mr of 49698. When the cloned gene was expressed in Staphylococcus carnosus, a 42 kDa protease was found in the culture medium. The protease was purified from both S. carnosus (pCAshp1) and S. hyicus subsp. hyicus. The N-terminal amino acid sequences of the two proteases revealed that ShpI is organized as a pre-pro-enzyme with a proposed 26 amino acid signal peptide, a 75 amino acid hydrophilic pro-region, and a 337 amino acid extracellular mature form with a calculated Mr of 38394. The N-termini showed microheterogeneity in both host strains. ShpI had a maximum proteolytic activity at 55°C and pH 7.4–8.5. The protease, which had a low substrate specificity, could be inhibited by metal- and zinc-specific inhibitors, such as EDTA and 1,10-phenanthroline. Insensitivity to phosphoramidon separates ShpI from the thermolysin-like family. The conserved Zn²⁺ binding motif, the only homology to other proteases, and the reactivation of the apoenzyme by Zn²⁺, indicated that Zn²⁺ is the catalytic ion. Ca²⁺ very probably acts as a stabilizer. We also demonstrated the presence of a second extracellular protease in S. hyicus subsp. hyicus.