共查询到20条相似文献,搜索用时 31 毫秒
1.
Chenyan Zhou Dongfeng Li Minchen Wu Wu Wang 《World journal of microbiology & biotechnology》2008,24(8):1393-1401
The xylanase gene xyn II from Aspergillus usamii E001 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K and integrated into the genome of a methylotrophic yeast, P. pastoris GS115, by electroporation. His+ transformants were screened for on the basis of their resistance to G418 and activity assay. A transformant, P. pastoris GSC12, which showed resistance to over 6 mg G418/ml and highest xylanase activity was selected. Recombinant xylanase was
secreted by P. pastoris GSC12 24 h after methanol induction of shake-flask cultures, and reached a final yield of 3139. About 68 U/mg 120 h after
the induction. The molecular mass of this xylanase was estimated to be 21 kDa by SDS-PAGE. The optimum pH and temperature
were 4.2 and 50 °C, respectively. Xylanase was stable below 50 °C and within pH 3.0–7.0. Its activity was increased by EDTA
and Co2+ ion and strongly inhibited by Mn2+, Li+ and Ag+ ions. The K
m and V
max values with birchwood xylan as the substrate were found to be 5.56 mg/ml and 216 μmol/mg/min, respectively. This is the first
report on expression and characterization of xylanase from A. usamii in P. pastoris. The hydrolysis products consisted of xylooligosaccharides together with a small amount of xylose. This property made the
enzyme attractive for industrial purposes, as relatively pure xylooligosaccharides could be obtained. 相似文献
2.
In recent years, the biotechnological use of xylanases has grown remarkably. To efficiently produce xylanase for food processing
and other industry, a codon-optimized recombinant xylanase gene from Streptomyces sp. S38 was synthesized and extracellularly expressed in Pichia pastoris under the control of AOX1 promoter. SDS-PAGE and activity assay demonstrated that the molecular mass of the recombinant xylanase was estimated to be
25 kDa, the optimum pH and optimum temperature were 5.5 and 50°C, respectively. In shake flask culture, the specific activity
of the xylanase activity was 5098.28 U/mg. The K
m
and V
max
values of recombinant xylanase were 11.0 mg/ml and 10000 μmol min−1 mg−1, respectively. In the presence of metal ions such as Ca2+, Cu2+, Cr3+ and K+, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of
Hg2+. This is the first report on the expression properties of a recombinant xylanase gene from the Streptomyces sp. S38 using Pichia pastoris. The attractive biochemical properties of the recombinant xylanase suggest that it may be a useful candidate for variety
of commercial applications. 相似文献
3.
Nguyen VN Oh IJ Kim YJ Kim KY Kim YC Park RD 《Journal of industrial microbiology & biotechnology》2009,36(2):195-203
Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography.
The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and
showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino
acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe
and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46
were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag+ and Hg2+ while Chi46 by Hg2+ and Pb2+ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co2+. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc)
n
, n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase. 相似文献
4.
The degradation of fluoroacetate by microorganisms has been established for some time, although only a handful of dehalogenases
capable of hydrolyzing the stable C–F bond have been studied. Pseudomonas fluorescens DSM 8341 was originally isolated from soil and readily degrades fluoroacetate, thus it was thought that its dehalogenase
might have some desirable properties. The enzyme was purified from cell-free extracts and characterised: it is a monomer of
32,500 Da, with a pH optimum of 8 and is stable between pH 4 and 10; its activity is stimulated by some metal ions (Mg2+, Mn2+ and Fe3+), but inhibited by others (Hg2+, Ag2+). The enzyme is specific for fluoroacetate, and the K
m
for this substrate (0.68 mM) is the lowest determined for enzymes of this type that have been investigated to date. 相似文献
5.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
6.
l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum
activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI
activity was stimulated by Mn2+, Fe3+, Fe2+, Ca2+ and inhibited by Cu2+, Ag+, Hg2+, Pb2+. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K
m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production
corresponds to a 39% equilibrium. 相似文献
7.
Hyaluronic acid production by recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> 总被引:1,自引:0,他引:1
A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would
be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at
25°C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml−1. The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified
enzyme is 6 and 40°C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The K
m and V
max values for birchwood xylan are 2.06 mg ml−1 and 0.49 mmol min−1mg−1, respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity
is unaffected by EDTA even at a concentration of 5 mM. 相似文献
8.
Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains
two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding
the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)6 tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric
protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration
up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the
hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature
and pH for the activity of the xylanase did not change. 相似文献
9.
Prabhu P Tiwari MK Jeya M Gunasekaran P Kim IW Lee JK 《Applied microbiology and biotechnology》2008,81(2):283-290
Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues
with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme
was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography,
suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn2+or Co2+, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k
cat of 12,455 min−1 and a k
cat/K
m of 34 min−1 mM−1 for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far. 相似文献
10.
S Canakcı Z Cevher K Inan M Tokgoz F Bahar M Kacagan FA Sal AO Belduz 《World journal of microbiology & biotechnology》2012,28(5):1981-1988
The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over
a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited
good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially
inhibited by Al3+ and Cu2+ but strongly inhibited by Hg2+. The enzyme follows Michaelis–Menten kinetics, with Km and Vmax values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan
in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose. 相似文献
11.
Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported
from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately
thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl2. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl2 were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium
by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H2S was detected in the headspace of cultures in the absence or presence of Hg2+, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have
potential for decontamination of solutions containing Hg2+ without production of toxic volatile H2S. 相似文献
12.
Qingxin Zhao Sheng Yuan Yuling Zhang Hong Zhu Chuanchao Dai Fang Yang Fengmin Han 《World journal of microbiology & biotechnology》2007,23(8):1057-1064
Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni2+-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml−1 medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn2+, Ca2+, Fe2+, Mg2+ and Fe3+ ions stimulated the pectate lyase activity, but Cu2+ and Zn2+ inhibited it. The recombinant PelA had a V
max of 77 μmol min−1 mg−1 and an apparent K
m of 0.50 mg ml−1 for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin
was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells. 相似文献
13.
A thermoalkaliphilic lipase of <Emphasis Type="Italic">Geobacillus</Emphasis> sp. T1 总被引:3,自引:0,他引:3
Leow TC Rahman RN Basri M Salleh AB 《Extremophiles : life under extreme conditions》2007,11(3):527-535
A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein
solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase
was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively.
The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had
an optimum temperature and pH of 70°C and pH 9, respectively. It was stable up to 65°C with a half-life of 5 h 15 min at pH
9. It was stable in the presence of 1 mM metal ions Na+, Ca2+, Mn2+, K+ and Mg2+ , but inhibited by Cu2+, Fe3+ and Zn2+. Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10–C14)
and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine
and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin.
The T
m for T1 lipase was around 72.2°C, as revealed by denatured protein analysis of CD spectra. 相似文献
14.
Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such
as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The
gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned
xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be
6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature
reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned
xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases
from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11. 相似文献
15.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献
16.
F. L. Zhang Z. M. Chi K. L. Zhu J. Li M. J. Li L. K. Liang L. F. Wu 《World journal of microbiology & biotechnology》2007,23(3):331-337
The full length empA gene encoding Vibrio anguillarum metalloprotease was amplified by PCR and fused to the expression vector pBAD24. The carboxy-terminal 6xHis-tagged recombinant
metalloprotein (rEmpA) was expressed from plasmid pBAD-VAP6his in E. coli TOP10 and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed a
molecular mass of the mature rEmpA predicted to be 36 kDa. The optimal temperature and pH for the purified rEmpA were 37°C
and 8.0, respectively. The enzyme was stable below 30°C and between pH 5.0 and 8.0, respectively. The results show that Ca2+, Na+ and Mg2+ had an activating effect on the enzyme while Zn2+ and Cu2+ acted as inhibitors of the enzyme. The purified rEmpA was characterized as a zinc metalloprotease as it was inhibited by
zinc- and metal-specific inhibitors, such as 1,10-phenanthroline, EDTA and EGTA. The results indicate that some characteristics
of EmpA from marine V. anguillarum had been modified after expression and processing in the engineered E. coli. The purified rEmpA showed degradation activity towards various kinds of proteins, indicating its potential role in pathogenesis. 相似文献
17.
Escherichia coli-expressed a hybrid xylanase, Btx, encoded by a designed hybrid xylanase gene btx was purified. The molecular mass of the enzyme was estimated to be 22 kDa. The K
m and k
cat values for Btx were 1.9 mg/ml and 140 s−1, respectively. It hydrolyzed xylan principally to xylobiose and xylotriose, and was functionally similar to family 11 xylanases.
As some differences were found in the hydrolytic products between birchwood xylan and wheat bran insoluble xylan, the xylan
binding domains in xylanase Btx must have different effects on soluble and insoluble xylan. 相似文献
18.
Cytochrome c
6
, (cyt c
6) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration
column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa
and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure
as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E
m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron
transfer processes is discussed. 相似文献
19.
Elaine O’Connell Patrick Murray Charles Piggott Franck Hennequart Maria Tuohy 《Journal of applied phycology》2008,20(5):557-565
A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative
electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity
studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate
methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (Km value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH
5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg2+, Co2+ and Fe3+. 相似文献
20.
Beris FS De Smet L Karaoglu H Canakci S Van Beeumen J Belduz AO 《Journal of microbiology (Seoul, Korea)》2011,49(4):641-650
The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be
classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment
was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range
(25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT
exhibited a low ATPase activity with K
m
− and V
max
− values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg2+ and Na+ ions, while Zn2+ and Al3+ stimulate the activity. Cd2+ and Ag+ reduced the activity and Li+, Cu2+, and Co2+ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the
activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily
and it can be responsible for aluminium tolerance in A. gonensis G2. 相似文献