Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326

Aspergillus CJ22-326, a fungi strain capable of utilizing chitosan as a carbon source, was isolated from soil samples. Two types of chitosanase (ChiA and ChiB) produced from the culture supernatant of Aspergillus CJ22-326 were purified to an apparent homogeneity identified by SDS–PAGE through ammonium sulfate precipitation, CM-Sepharose FF chromatography, and Sephacryl S-200 gel filtration. Molecular weights of the enzymes were 109 kDa (ChiA) and 29 kDa (ChiB). Optimum pH values and temperature of ChiA were 4.0 and 50 °C, respectively, those of ChiB were 6.0 and 65 °C. The enzyme activities of ChiA and ChiB were increased by about 0.5-fold and 1.5-fold, respectively, by the addition of 1 mM Mn²⁺. However, 2.5 mM Ag⁺, Hg²⁺ and Fe³⁺strongly inhibited ChiA and ChiB activities. Viscosimetric assay and analysis of reaction products of these enzymes, using chitosan as a substrate, by TLC indicated endo- and exo-type cleavage of chitosan by ChiB and ChiA, respectively. ChiB catalysed the hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, and chitosan with a low degree of acetylation (0–30%), and formed chitotriose with chitohexaose as the major products. ChiA released a single glucosamine residue from chitosan and glucosamine oligomers. Both of the activities of ChiA and ChiB increased with the degree of deacetylation of chitosan. The enzyme ChiB had a useful reactivity and a high specific activity for producing functional chitooligosaccharides with high degree of polymerization.     

Biochemical and genetic characterization of a D(-)-3-hydroxybutyrate dehydrogenase from Acidovorax sp. strain SA1

D(-)-3-hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) from a poly(D(-)-3-hydroxybutyrate) (PHB) degrading bacterium, Acidovorax sp. SA1, was purified using Toyopearl DEAE-650M, red-Sepharose CL-4B, and Q Sepharose FF. The molecular mass of the enzyme was estimated as 27 kDa by SDS-PAGE and 110 kDa by gel filtration. The gene encoding BDH was cloned and sequenced, and expressed in Escherichia coli. The gene product was purified in two steps with a high yield. The N-terminal amino acid sequence of the enzyme purified from E. coli agreed with that of the purified enzyme from strain SA1. The BDH of strain SA1 had high amino acid sequence homology to that of Ralstonia eutropha H16. The Km values for D(-)-3-hydroxybutyrate and NAD+ in the oxidation reaction were 4.5 x 10(-4) M and 8.9 x 10(-5) M, respectively. The Km values for acetoacetate and NADH in the reduction reaction were 2.4 x 10(-4) M and 2.9 x 10(-5) M, respectively.     

Microbial demetallization of crude oil: nickel protoporphyrin disodium as a model organo-metallic substrate

A soil isolate designated as YA-1 strain was selected for its ability to degrade nickel protoporphyrin disodium (NiPPDS). The strain was capable of utilizing NiPPDS as the sole source of carbon. This strain, a gram-negative aerobic rod, was identified as Pseudomonas azelaica YA-1 based on the result of its 16S rRNA analysis. Product analyses by HPLC showed that this strain can decompose the porphyrin ring to which a metal ion is bound. However, the use of whole bacterial cells cannot result in extensive NiPPDS degradation; therefore, the YA-1 enzyme was extracted and purified. This NiPPDS-degrading enzyme named as protoporphyrinase was purified from P. azelaica YA-1 by ammonium sulfate fractionation and sequential chromatographies using DEAE Toyopearl 650 M, CM Toyopearl 650 M and Biogel P-60 columns, with a yield of 11.3% based on the enzyme activity and an overall purification of 498-fold. The molecular weight of this enzyme is estimated to be 39,000 Da by SDS-PAGE and 34,000 Da by gel filtration. The optimum pH and temperature for the enzyme were 7.0 and 30 degrees C, respectively. The activity was stable at pH 2.0-11.0 and at temperatures below 50 degrees C. The enzyme activity was inactivated by ferric chloride, potassium ferricyanide, ZnCl2 and CdCl2.     

Purification, bacteriolytic activity, and specificity of beta-lytic protease from Lysobacter sp. IB-9374

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.     

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.     

Disintegration of uncooked rice by carboxymethyl cellulase from Sporotrichum sp. HG-I

A thermostable carboxymethyl cellulase (CMCase) was purified to homogeneity from a wheat bran culture of the thermophilic fungus Sporotrichum sp. HG-1, an isolate from a compost heap. The enzyme had a molecular weight (M(r)) of 33,000 by SDS-PAGE. The optimum temperature and pH for enzyme activity were 70 degrees C and 4.5-5.0, and the enzyme was heat stable. Uncooked Thai rice was digested so as to cause its disintegration by the addition of purified CMCase, but not by the addition of xylanase purified from strain HG-1. Ferulic acid conjugated to oligosaccharide was released significantly by the combined action of CMCase and xylanase, but the free form of ferulic acid was not detectable.     

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0?7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca ²⁺ , Mn²⁺, PCMB, and DTNB, but inhibited by Ba²⁺, Fe³⁺, Hg²⁺, Mg²⁺, and iodoacetic acid. The purified enzyme was of α?-type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α?-1,4-linkages and with increasing percentage of α?-1,6-linkages in substrate similar to the results from commercially available honey.     

Enzymatic degradation of cellulose acetate plastic by Novel degrading bacterium Bacillus sp. S2055

Cellulose acetate (CA)-degrading bacteria were isolated from samples obtained from environments at a population size of 6.7 x 10(1) to 1.0 x 10(8) halo-forming cfu/ml-water or g-solid, suggesting their ubiquitous presence. The classification of 35 isolated strains of CA-degrading bacteria into 15 genera indicates that CA-degrading activity is over a wide range of taxonomical groups. From these isolates, Bacillus sp. S2055 was found to be the most efficient CA-degrading bacterium, and its CA-degrading enzyme(s) was partially characterized. The weight loss of CA plastic film (degree of substitution (DS)=1.7) in the culture of S2055 was less than 12% after a 35-d culture while that in the crude enzyme solution extracted from the culture supernatant reached 62% after the same period. Lipase and cellulase activities were detected in the culture supernatant of strain S2055. The crude enzyme solution contained three major protein fractions that have different mean molecular weights (MWs). Fraction I with the highest MW exhibited both lipase and cellulase activities, while fraction II and III exhibited only lipase activity. Fraction I significantly deacetylated CA (DS 1.5) and fragmented CA plastic film into pieces while the other fractions failed to do so even when used in combination with commercially-available cellulases and lipases. The lipase activity of fraction I against various substrates differed considerably from those of known lipases. It was thus suggested that deacetylation of CA mediated by an enzyme with such a peculiar lipase-like activity is a requisite for the efficient biodegradation of CA plastics.     

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.     

Degradation of cell wall materials from sweetpotato, cassava, and potato by a bacterial protopectinase and terminal sugar analysis of the resulting solubilized products

Cell wall materials (CWMs) from sweetpotato, cassava, and potato starch residues were degraded using a crude enzyme solution from the culture filtrate of a Bacillus sp. isolated from soil, Bacillus sp. M4. This organism has been found to secrete polygalacturonic acid lyase (PGL) and glycan depolymerase activities, especially arabinanase, but cellulase activity was nearly absent. Sugar analysis of the solubilized product after enzyme treatment at pH 7.0 revealed that it is mainly composed of galacturonic acid, galactose, and arabinose, the sugars found commonly in the pectin fraction. This suggested the presence of a protopectinase (PPase) activity in the culture filtrate. The presence of EDTA completely inhibited PGL but PPase activity was almost retained, suggesting that the PGL is not the primary activity responsible for pectin solubilization. The mode of action of the crude enzyme was determined by terminal sugar analysis using HPAEC-PAD after hydrolysis of the reduced products. Results revealed that galactose is the main neutral sugar at the reducing terminal of the products, although rhamnose was also present in the higher molecular weight component. This suggested that at neutral pH, the primary activity in the culture filtrate of Bacillus sp. M4 is a B-type PPase, which attacked the galactan as well as rhamnogalacturonan moieties of the protopectin, resulting in the release of a soluble pectin fraction.     

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.     

胞外弹性蛋白酶的理化特性及其影响因素

分离得到的假单胞菌(Pseudomonas sp) 菌株具有较强的分泌胞外弹性蛋白酶的能力。经微生物发酵方法生产的弹性蛋白酶,经过盐析、透析、DEAE SephadexA2 5离子交换层析、SephadexG75凝胶过滤层析等纯化步骤,从其发酵液中得到了均一的酶制品。研究结果表明,酶的最适作用温度为5 0℃;在硼砂-硼酸缓冲液中最适作用pH为8 0左右;酶在碱性环境下( pH7 0～12 0 )稳定性较好;在37℃以下,酶的稳定性较高。超过6 0℃,酶在短时间内即失活     

PP-R, 7-(2-hydroxyethyl)-monascorubramine, a red pigment produced in the mycelia of Penicillium sp. AZ

A red pigment was extracted and purified from the mycelia of a newly isolated strain of Penicillium sp., Penicillium sp. AZ, which produced a soluble violet pigment PP-V, 12-carboxyl-monascorubramine, in the culture fluid. The red pigment, PP-R, was determined by FAB-MS and 1H and 13C NMR to be a novel compound, 7-(2-hydroxyethyl)-monascorubramine.     

北京棒杆菌天冬氨酸激酶G377定点突变及酶学性质表征

采用饱和定点突变和高通量筛选技术,对北京棒杆菌天冬氨酸激酶（aspartate kinase,AK）AK Gly377位点进行突变,并筛选获得高活力突变体G377F,分离纯化后对G377F AK酶学性质进行表征。结果表明：突变体AK最适pH值为9.0,较突变前野生型（wide type,WT）最适pH值为8.5有所提高;最适反应温度与WT一致,均为25 ℃;半衰期由WT的2.6 h提高到5.3 h;pH耐受性在5 h内保持其活力50%以上,与WT 3 h内酶活力保持能力相同。G377F对金属离子和有机溶剂均表现出良好的抗性。其中,Lys的抑制作用在一定程度上被解除,当Lys和Met同时存在时对AK具有明显的激活作用。动力学研究显示：G377F AK Vmax较WT提高9.3 倍;n值为1.0明显低于WT的2.7,说明AK正协同性降低,趋向于米氏酶。     

一株褐藻酸裂解酶高产菌的筛选及其酶学性质研究

从腐烂的海带中筛选出一株褐藻酸裂解酶（Aly）高产菌,经形态学分析、生理生化特征和分子水平鉴定,该菌为交替假单胞菌属,命名为Pseudoalteromonas sp. EE258。利用硫酸铵沉淀、快流速DEAE-琼脂糖凝胶（DEAE-sepharose Fast Flow）和丙烯葡聚糖凝胶（Sephacryl S-100）柱层析等方法,对Pseudoalteromonas sp. EE258产生的褐藻酸裂解酶Aly-EE258进行分离纯化,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定该酶的分子质量为23 ku。酶学性质研究显示：该酶的最适反应温度为40 ℃,最适反应pH值为7.0,在20 ℃以下时稳定性较好,Ca2+和Ba2+能明显提高酶的活性,Fe2+、Al3+、Mg2+和乙二胺四乙酸（EDTA）抑制酶的活性。Aly-EE258能不同程度地裂解褐藻酸、聚古洛糖醛酸和聚甘露糖醛酸,其中聚甘露糖醛酸裂解后主要产生单一的低聚寡糖,在实际应用方面具有重要意义。     

酶法改变甘草苷糖醛酸基提高其甜度的研究(Ⅱ)——能水解甘草苷糖醛酸基酶的提纯与酶性质研究

以酶法改变甘草苷糖醛酸基提高其甜度为目的,对新分离筛选的甘Ｍ－２和甘Ｍ－６霉菌产的β－葡萄糖醛酸苷酶进行了分离提纯和酶学方面的研究。结果表明,两株菌产的β－葡萄糖醛酸苷酶被６０％饱和硫酸铵沉淀较好,经ＤＥＡＥ－ＣｅｌｕｏｓｅＤＥ５２离子交换层析柱梯度洗脱,得到纯酶,提纯倍数分别为１０．６７和６．１５倍,收率分别为３３．０％和２４．２％,其中甘Ｍ－２菌产β－葡萄糖醛酸苷酶只能将甘草苷水解成甘草次酸（ＧＡ）;甘Ｍ－６菌产β－葡萄糖醛酸苷酶水解甘草苷主要变成甜度很高的单葡萄糖醛酸基甘草苷（ＧＡＭＧ）;两种酶在ＳＤＳ－聚丙烯酰胺凝胶电泳都得到电泳单点,其酶蛋白相对分子质量分别为６００００和４２０００;酶反应最适ｐＨ分别为５．０和６．０,最适温度均为４０℃,均在ｐＨ４．０～８．０和２０～７０℃范围内相对稳定。     

嗜热拟青霉木糖苷酶的性质及其与木聚糖酶的协同作用

嗜热拟青霉(Paecilomyces thermophila)J18利用玉米芯能产胞外木糖苷酶,通过(NH_4)_2SO_4沉淀、DEAE-52离子交换层析及Q-琼脂糖凝肢-FF(QSFF)离子交换层析从上清液纯化得到了电泳纯的木糖苷酶,纯化倍数为31.9倍,回收率为2.27%。SDS-PAG E及Superdex-75凝胶过滤层析测定木糖苷酶的分子量分别为53.5 ku和51.8 ku。该酶最适温度及pH分别为55℃和pH 6.5。木糖苷酶能够水解木二糖和低聚木糖但不能水解木聚糖,水解低聚木糖的相对速率随聚合度增加而增加。该酶对木糖的抑制常数Ki值为139 mmol/L,具有很高的木糖耐受性。木糖苷酶与内源木聚糖酶一起水解木聚糖产生更多的还原糖,表现出协同作用。     

木薯渣中乳酸菌的分离鉴定及其产酸能力分析

该研究利用传统培养法从自然发酵木薯渣中分离得到9株菌,通过菌株形态、生理生化特征及16S rDNA基因序列分析方法对菌株进行鉴定。结果显示,这9株菌均为乳杆菌属（Lactobacillus sp.）,其中菌株Y5、Y13、M14为干酪乳杆菌（Lactobacillus casei）,菌株Y6、Y12、M12为植物乳杆菌（Lactobacillus plantarum）,菌株Y7为丘状乳杆菌（Lactobacillus collinoides）,菌株M9为副干酪乳杆菌（Lactobacillus paracasei）,菌株M11为类布氏乳杆菌（Lactobacillus parabuchneri）;产酸及耐酸试验结果表明这5种菌均具有较强的产酸能力及耐酸性,发酵6 h后发酵液中pH值可达4.5以下,其中菌株Y12和M14的产酸能力最强,在15 h后其pH值均在3.6以下,可用于木薯渣或其他青贮饲料发酵剂的研制。     

Ytterbium-decreasing Streptomyces sp. and its naphthoquinone-pigment production in the presence of rare-earth elements

We screened for oligotrophic microorganisms capable of decreasing the concentration of ytterbium (Yb), a representative of the heavy rare-earth elements, in a culture medium. From 476 strains of oligotrophic microorganisms (grown on 1/100 diluted nutrient agar) isolated from soil and river water samples, 5 strains capable of reducing the concentration of Yb in diluted nutrient broth containing 5 ppm Yb were selected. Among them, a strain capable of reducing the concentration of all rare-earth elements to a great extent was identified as Streptomyces sp. (strain YB-1). This strain produced redish-purple pigment(s) only in the presence of rare earths, but not in that of other metals. The pigment was extracted with ethyl acetate and purified by a series of column chromatography steps. From the results of structural analysis using ultraviolet or infrared absorption spectrometry and 13C-NMR, the pigment was determined to be a kind of naphthoquinone similar to nanaomycin produced by a Streptomyces sp. These results suggested that rare earths might affect the physiological activity of this strain.