Purification and properties of an extracellular inulinase from Rhizopus sp. strain TN-96

A filamentous fungus, Rhizopus sp. strain TN-96, was isolated from rhizosphere soil samples. An extracellular inulinase was purified from the culture filtrate of strain TN-96 grown on inulin by DEAE-Cellulofine A-500 and Sephacryl S-200 HP chromatographies. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M(r) of 83 kDa. The purified enzyme had specific activities of 17 U/mg toward inulin (I) and 0.32 U/mg toward sucrose (S) (I/S ratio, 53). Inulinase activity was optimal at pH 5.5 and 40 degrees C. The inulinase exhibited an apparent K(m) value of 9.0 mM for inulin. The enzyme also hydrolyzed raffinose, but not bacterial levan.     

Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506

Phosphite dehydrogenase (PtxD), which catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH, has great potential for NADH regeneration in industrial biocatalysts. Here, we isolated a soil bacterium, Ralstonia sp. strain 4506, that grew at 45°C on a minimal medium containing phosphite as the sole source of phosphorus. A recombinant PtxD of Ralstonia sp. (PtxD(R4506)) appeared in the soluble fraction in Escherichia coli. The purified PtxD(R4506) showed 6.7-fold greater catalytic efficiency (V(max)/K(m)) than the first characterized PtxD of Pseudomonas stutzeri (PtxD(PS)). Moreover, the purified PtxD(R4506) showed maximum activity at 50°C, and its half-life of thermal inactivation at 45°C was 80.5h, which is approximately 3,450-fold greater than that of PtxD(PS). Therefore, we concluded that PtxD(R4506), which shows high catalytic efficiency, solubility, and thermostability, would be useful for NADH regeneration applications.     

青霉菌B01 产菊粉酶特性的研究及菊粉酶系分析

研究了pH 值、温度、底物浓度、金属离子对青霉菌(Penicillium sp.)B01 所产胞外菊粉酶活性的影响以及酶活随反应时间的变化情况。结果表明,pH4.6 时菊粉酶活力最大,最适反应温度是55℃。在pH3.6～5.4 的范围内,60℃以下酶活较稳定。Ca2+ 对菊粉酶有激活作用,而Cu2+ 对其活性强烈抑制。采用活性聚丙烯酰胺凝胶电泳法(PAGE)对该酶粗液进行蛋白质分析,获得较清晰的6 条蛋白谱带;2,3,5- 氯化三苯基四氮唑(TTC)活性染色结果显示,只有3 条谱带表现为具有菊粉酶活性。对酶解产物用薄层层析(TLC)和高效液相色谱(HPLC)法的分析结果,进一步证实这3 条谱带具有菊粉酶活性,并确定其主要为外切型菊粉酶,产物主要为果糖。     

Production and Application of Xylanase from Penicillium sp. Utilizing Coffee By-products

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K _m 5.6 mg/mL, and V _max 925 μmol mg⁻¹ min⁻¹ with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.     

Purification and characterization of an extracellular beta-agarase from Bacillus sp. MK03

A new beta-agarase was purified from an agarolytic bacterium, Bacillus sp. MK03. The enzyme was purified 129-fold from the culture supernatant by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The purified enzyme appeared as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Estimation of the molecular mass by SDS-PAGE and gel filtration gave values of 92 kDa and 113 kDa, respectively. The N-terminal amino acid sequence of the enzyme showed no homology to those of other known agarases. The optimum pH and temperature for this enzyme were 7.6 and 40 degrees C, respectively. The predominant hydrolysis product of agarose by this enzyme was neoagarotetraose, indicating the cleavage of beta-1,4 linkage. This enzyme could hydrolyze neoagarohexaose to produce neoagarotetraose and neoagarobiose; it could not hydrolyze these products. The enzyme digested agarose by endo-type hydrolysis.     

Purification and characterization of an epsilon-poly-L-lysine-degrading enzyme from the epsilon-poly-L-lysine-tolerant Chryseobacterium sp. OJ7

The epsilon-poly-L-lysine-degrading enzyme of the epsilon-poly-L-lysine-tolerant Chryseobacterium sp. OJ7 was purified and characterized. The bacterium excreted the enzyme into the culture filtrate. The purified enzyme has a molecular mass of approximately 38.4 kDa and consists of two identical subunits with a molecular mass of 19.5 kDa. The enzyme catalyzed the endo-type degradation of epsilon-poly-L-lysine. A correlation between the epsilon-poly-L-lysine tolerance of the bacterium and the high epsilon-poly-L-lysine-degrading activity was suggested.     

Purification and characterization of an l-amino acid oxidase from Pseudomonas sp. AIU 813

An l-amino acid oxidase was found from a newly isolated strain, Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with l-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with l-lysine as ligand. The enzyme oxidized l-lysine, l-ornithine and l-arginine, but not other l-amino acids and d-amino acids. The oxidase activity for l-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for l-ornithine and l-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a flavoprotein and composed of two identical subunits with molecular mass of 54.5?kDa. The N-terminal amino acid sequence was similar to that of putative flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of l-amino acid oxidases.     

Purification and characterization of an extracellular alpha-neoagarooligosaccharide hydrolase from Bacillus sp. MK03

An agarolytic bacterium was isolated from soil in Gifu prefecture, Japan, and identified as Bacillus sp. strain MK03. The strain secreted neoagarooligosaccharide hydroluse into the culture medium. The enzyme was purified 49.7-fold from the culture fluid by ammonium sulfate precipitation and anion-exchange and gel-filtration column chromatographic methods. The purified enzyme appeared as a single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. Estimations of the molecular mass by gel filtration and SDS-PAGE gave values of 320 kDa and 42 kDa, respectively, indicating that the enzyme is octametric. The enzyme cleaved the alpha-1,3 linkage in neoagarobiose to produce 3,6-anhydro-L-galactose and D-galactose. It also selectively cleaved the alpha-1,3 linkage at the nonreducing end in neoagarotetraose or neoagarohexaose to give 3,6-anhydro-L-galactose and agarotriose or agaropentaose. The optimum temperature and pH for the enzyme were 30 degrees C and 6.1, respectively. The N-terminal amino acid sequence showed no homology to sequences of other known neoagarooligosaccharide hydrolases and agarases.     

Streptomyces lavendulae X33海藻糖合酶基因克隆及酶学特性

目的：对菌株Bacillus sp. B110的胞内麦芽糖淀粉酶BMAL进行基因克隆、异源表达、纯化及酶学性质研究,为后期开发新的淀粉加工用酶打下基础。方法：使用PCR技术对Bacillus sp. B110的胞内麦芽糖淀粉酶bmal基因序列进行全长克隆,异源表达,使用Ni²⁺-NTA进行纯化,再对其酶学特性进行测定,使用序列分析工具BioEdit、MEGA等对其氨基酸序列进行分析,使用AlphaFold2对其三级结构进行预测分析。结果：BMAL基因全长1770 bp,编码一个589氨基酸残基的蛋白。重组酶rBMAL经Ni²⁺-NTA亲和层析纯化后,SDS-PAGE电泳结果显示其分子量大小为63 kDa。氨基酸序列分析和三维建模表明BMAL与来源于B.subtilis 168和B.subtilis SUH4-2的麦芽糖淀粉酶有较高的一致性,且BMAL具有一个麦芽糖淀粉酶所独有的N端结构域以及由Asp328-Glu357-Asp424三个氨基酸残基所构成的催化中心。重组酶rBMAL最适反应温度为45 ℃,最适反应pH为6.0。重组酶rBMAL在30 ℃条件下保藏7 h残留酶活为60%,但在60 ℃条件下保藏2 h残留酶活力下降98%,说明BMAL对热敏感。重组酶rBMAL在4 ℃,pH7.0~9.5保藏12 h活性稳定。当存在1 mmol/L的金属离子Mg²⁺时,重组酶rBMAL活力提高36%,而Ni²⁺、Fe³⁺、Co²⁺、Cu²⁺、Zn²⁺、Al³⁺、Ca²⁺对重组酶rBMAL有抑制作用,酶活力减少85%~48%。有机溶剂和化学试剂甲醇、乙醇、丙酮、异腈、EDTA和SDS对重组酶rBMAL有较强的抑制作用,酶活力减少至32.3%~64.8%。底物特异性实验结果证实BMAL最适底物为环精糊。结论：Bacillus sp. B110的胞内麦芽糖淀粉酶BMAL具有良好的催化特性和pH稳定性,在面包烘焙工业上具有潜在的应用价值。     

海洋青霉碱性脂肪酶液态发酵和部分酶学性质研究

从海洋菌中分离筛选到1株碱性脂肪酶产生菌H-19,对该菌产脂肪酶的液态发酵条件进行了优化,优化后的培养基组成(%):葡萄糖0.5,大豆油1.0,蛋白胨1.0,(NH4)2SO40.5,NaCl 2.75,KCl 0.1,MgCl20.5,MgSO4.7H2O 0.2,CaCl20.05;发酵培养基起始pH8.0,发酵温度28℃,摇床转速180 r/min,发酵周期48 h,脂肪酶的活力可达17.43 IU/mL。酶的最适反应pH为9.4,最适温度36℃,半失活温度为60℃。     

南海深海链霉菌Streptomyces sp.SCSIO 13580脂肪酶的酶学性质

对从南海深海链霉菌Streptomyces sp.SCSIO 13580中的新颖脂肪酶L-1进行了酶学性质鉴定。脂肪酶L-1对对硝基苯酚己酸酯的催化活性最高,最适反应温度为40℃,最适反应p H为8.0,在最适条件下的酶活力为51.5 U/mg。脂肪酶L-1对大部分有机溶剂和表面活性剂具有极好的耐受性。同时该南海深海微生物脂肪酶能在温和条件下催化制备多种短链酯食品香料产品,具有较好的工业应用前景。     

菊粉外切酶的异源表达、纯化及酶学性质

将菊粉降解菌Paenibacillus sp. Lfos16的菊粉外切酶基因克隆至表达载体p ET-28a (+),转入Escherichia. coli BL21(DE3)中实现了异源表达。利用镍柱亲和层析纯化重组菊粉外切酶,并进行SDS-PAGE检测。重组菊粉外切酶的表观分子质量为87 k Da,经纯化后,重组菊粉外切酶的比酶活为348.30 U/mg。重组菊粉外切酶作用菊粉和蔗糖的酶活分别为259.37 U/m L和592.16 U/m L,I/S值为0.438,且重组酶水解菊粉的主要产物为果糖。重组菊粉外切酶最适作用温度为40℃,且当温度低于30℃时酶活较稳定;最适作用pH为6。Ag~+、Cu~(2+)、Mn~(2+)、Zn~(2+)、Hg~(2+)、Fe~(3+)具有显著抑制作用。重组菊粉外切酶对菊粉的K_m为19. 28 mg/m L,Vmax为0.18 mg/(min·m L)。     

Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719

An alkaline protease was purified to apparent homogeneity from culture supernatants of Bacillus sp. PS719, a novel alkaliphilic, thermophilic bacterium isolated from a thermal spring soil sample, by ammonium sulfate precipitation followed by DEAE-cellulose and alpha-casein agarose column chromatographies. The purified enzyme migrated as a single protein band of 42 kDa during both denaturing and nondenaturing gel electrophoresis, suggesting that it consists of a single polypeptide chain. Its isoelectric point was approximately 4.8. The protease exhibited maximum activity towards azocasein at pH 9.0 and at 75 degrees C. The enzyme activity was stimulated by Ca2+, but was inhibited in the presence of Fe2+ or Cu2+. The enzyme was stable in the pH range 8.0 to 10.0 and up to 80 degrees C in the absence of Ca2+. Since phenylmethylsulfonyl fluoride (PMSF) and 3,4-dichloroisocoumarin (DCI) in addition to N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) completely inhibited the activity, this enzyme appears to be a trypsin-like serine protease. Among the various oligopeptidyl-p-nitroanilides tested, the protease showed a preference for cleavage at arginine residues on the carboxylic side of the scissile bond of the substrate, liberating p-nitroaniline from N-carbobenzoxy (CBZ)-L-arginine-p-nitroanilide with the K(m) and V(max) values of 0.6 mM and 1.0 micromol.min(-1).mg protein(-1), respectively.     

Contribution of Penicillium sp. to the Flavors of Brie and Camembert Cheese

Volatile flavor compounds produced by Penicillium caseicolum in domestic and French Brie cheese were identified by gas chromatography-mass spectrometry; they included carbon-5, carbon-7, Carbon-9, carbon-11 methyl ketones, and corresponding secondary alcohols, 2-octanone, 3-octanone, 1-octen-3-ol, 1,5-octadien-3-ol, 1,5-octadien-3-one, and 2-methylisoborneol. Lipoxygenase formation of the eight carbon compounds responsible for the mushroom, plant-like flavors of mold surface-ripened cheeses was enhanced either by the disruption of mold mycelia or the addition of specific ω-3 and ω-6 fatty acid precursors. The occurrence of 8-nonen-2-one in mold-ripened cheeses was associated with the β-oxidative conversion of a specific unsaturated fatty acid resulting from initial lipoxygenase attack on either linoleic or linolenic acids.     

Purification and characterization of dibenzothiophene sulfone monooxygenase and FMN-dependent NADH oxidoreductase from the thermophilic bacterium Paenibacillus sp. strain A11-2

A dibenzothiophene (DBT) sulfone monooxygenase (TdsA), which catalyses the oxidative CS bond cleavage of DBT sulfone to produce 2-(2-hydroxyphenyl)benzenesulfinate (HPBS) was purified from the thermophilic DBT desulfurizing bacterium Paenibacillus sp. strain A11-2 by multistep chromatography. The molecular mass of the purified enzyme was determined to be 120 kDa by gel filtration and the subunit molecular mass was calculated to be 48 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicating a dimeric structure. The N-terminal amino acid sequence of the purified TdsA was determined to be MRQMHLAGFFAAGNTHH, which revealed no significant similarity to any other known amino acid sequences. The purified TdsA absolutely required an oxidoreductase for its activity. This oxidoreductase (TdsD) was also purified to homogeneity, and its molecular size was calculated to be 50 kDa and 25 kDa by gel filtration and SDS-PAGE, respectively. TdsD was completely FMN-dependent, and FAD could not act as a cofactor. The N-terminal amino acid sequence of the purified TdsD was determined to be TSQTAEQSIAPIVAQYRHPEQPISALFVNR, which showed significant similarity to kinesin-like protein (44% identity). The optimal temperatures for the activity of TdsA and TdsD were 45 degrees C and 55 degrees C, respectively. Both enzymes showed optimal activity at pH 5.5. TdsA was slightly inhibited by sulfate, but not by 2-hydroxybiphenyl (2-HBP), which is another end product of DBT. TdsA showed higher activity toward bulkier substrates than its mesophilic counterpart, DszA. These properties suggest the applicability of biodesulfurization to the processing of actual petroleum fractions.     

Purification and characterization of a novel beta-agarase from an alkalophilic bacterium, Alteromonas sp. E-1

A novel beta-agarase (EC 3.2.1.81) was purified from an agar-degrading alkalophilic bacterium, Alteromonas sp. E-1 isolated from the soil. This enzyme was obtained from a cell-free extract after sonication and purified 40.9-fold through treatment with streptomycin, ammonium sulfate fractionation and successive chromatography on anion-exchange and gel filtration columns. The molecular weight was estimated to be 82 kDa by SDS-polyacrylamide gel electrophoresis and 180 kDa by Superdex 200 gel filtration. The enzyme was inhibited by Mn2+, Cu2+, Fe2+, Zn2+ and Hg2+, and activated by K+, Na+ and EDTA, and its optimum pH and temperature for agarose degradation were 7.5 and 40 degrees C, respectively. This beta-agarase hydrolyzed agarose with rapid reduction of viscosity, and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose] was detected from the early stage of the reaction. Neoagarobiose as the final product was selectively released from agarose, neoagarohexaose and neoagarotetraose by the reaction with this beta-agarase. This observation was different from that of other beta-agarases which produced mixtures of neoagarobiose and neoagarotetraose as the final hydrolysis products. The N-terminal amino acid sequence of this beta-agarase shows no homology to those of other beta-agarases that were so far reported.