scholarly journals Improved Production of Xylanase in Pichia pastoris and Its Application in Xylose Production From Xylan

2021 ◽  
Vol 9 ◽  
Author(s):  
Ting Miao ◽  
Abdul Basit ◽  
Junquan Liu ◽  
Fengzhen Zheng ◽  
Kashif Rahim ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cost Effective ◽  
Biomass Energy ◽  
Peptide Engineering ◽  
Xylanolytic Enzyme ◽  
Xylose Production ◽  
Hydrolysis Of ◽  
Maximal Production

Xylanases with high specific activity has been focused with great interest as a useful enzyme in biomass utilization. The production of recombinant GH11 xylanase (MYCTH_56237) from Myceliophthora thermophila has been improved through N-terminal signal peptide engineering in P. pastoris. The production of newly recombinant xylanase (termed Mtxyn11C) was improved from 442.53 to 490.7 U/mL, through a replacement of α-factor signal peptide with the native xylanase signal peptide segment (MVSVKAVLLLGAAGTTLA) in P. pastoris. Scaling up of Mtxyn11C production in a 7.5 L fermentor was improved to the maximal production rate of 2503 U/mL. In this study, the degradation efficiency of Mtxyn11C was further examined. Analysis of the hydrolytic mode of action towards the birchwood xylan (BWX) revealed that Mtxyn11C was clearly more effective than commercial xylanase and degrades xylan into xylooligosaccharides (xylobiose, xylotriose, xylotetraose). More importantly, Mtxyn11C in combination with a single multifunctional xylanolytic enzyme, improved the hydrolysis of BWX into single xylose by 40%. Altogether, this study provided strategies for improved production of xylanase together with rapid conversion of xylose from BWX, which provides sustainable, cost-effective and environmental friendly approaches to produce xylose/XOSs for biomass energy or biofuels production.

scholarly journals Preliminary X-ray diffraction analysis of a thermophilic β-1,3–1,4-glucanase fromClostridium thermocellum

2014 ◽  
Vol 70 (7) ◽  
pp. 946-948 ◽  
Author(s):  
Lilan Zhang ◽  
Puya Zhao ◽  
Chun-Chi Chen ◽  
Chun-Hsiang Huang ◽  
Tzu-Ping Ko ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
High Specific Activity ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Glycosidic Bonds ◽  
Hydrolysis Of

β-1,3–1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3-O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A fromClostridium thermocellumexhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed inEscherichia coli, purified and crystallized in the trigonal space groupP3121, with unit-cell parametersa=b= 74.5,c= 182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress.

Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

1977 ◽  
Vol 37 (03) ◽  
pp. 556-565 ◽  
Author(s):  
S. E Papaioannou ◽  
W. J Marsheck
Keyword(s):  
Methyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Soil Bacterium ◽  
Ester Hydrochloride ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Hydrolysis Of

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

scholarly journals Identification of an Invertase With High Specific Activity for Raffinose Hydrolysis and Its Application in Soymilk Treatment

2021 ◽  
Vol 12 ◽  
Author(s):  
Juanjuan Liu ◽  
Jing Cheng ◽  
Min Huang ◽  
Chen Shen ◽  
Ke Xu ◽  
...  
Keyword(s):  
Nutritional Value ◽  
Specific Activity ◽  
High Specific Activity ◽  
Maximum Activity ◽  
Beneficial Bacteria ◽  
Human Gut ◽  
Mild Conditions ◽  
Alternative Approach ◽  
Hydrolysis Of

The hydrolyzation of raffinose into melibiose by using invertases under mild conditions improves the nutritional value of soybean products. However, this strategy has received little attention because a suitable invertase remains lacking. In this study, a novel invertase named InvDz13 was screened and purified from Microbacterium trichothecenolyticum and characterized. InvDz13 was one of the invertases with the highest specific activity toward raffinose. Specifically, it had a specific activity of 229 U/mg toward raffinose at pH 6.5 and 35°C. InvDz13 retained more than 80% of its maximum activity at pH 5.5–7.5 and 25–40°C and was resistant to or stimulated by most cations that presented in soymilk. In soymilk treated with InvDz13 under mild conditions, melibiose concentration increased from 3.1 ± 0.2 to 6.1 ± 0.1 mM due to raffinose hydrolyzation by InvDz13. Furthermore, the prebiotic property of InvDz13-treated soymilk was investigated via in vitro fermentation by human gut microbiota. Results showed that InvDz13 treatment increased the proportion of the beneficial bacteria Bifidobacterium and Lactobacillus by 1.6- and 3.7-fold, respectively. By contrast, the populations of Escherichia and Collinsella decreased by 1.8- and 11.7-fold, respectively. Thus, our results proved that the enzymatic hydrolysis of raffinose in soymilk with InvDz13 was practicable and might be an alternative approach to improving the nutritional value of soymilk.

scholarly journals Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2005 ◽  
Vol 187 (6) ◽  
pp. 2077-2083 ◽  
Author(s):  
Sherry V. Story ◽  
Claudia Shah ◽  
Francis E. Jenney ◽  
Michael W. W. Adams
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
High Specific Activity ◽  
Native Form ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

scholarly journals The Incorporation of Leucine-C14 into Microsomal Particles and other Subcellular Components of the Pea Epicotyl

1959 ◽  
Vol 5 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Paul O. P. Ts'o ◽  
Clifford S. Sato
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
High Specific Activity ◽  
Insoluble Fraction ◽  
Supernatant Fraction ◽  
Group Method ◽  
Pea Seedlings ◽  
Pea Epicotyl ◽  
Hydrolysis Of ◽  
End Group

Incorporation of leucine-C14 into subcellular fractions of the apical section of pea seedlings has been studied as a function of the length of incubation. The specific activity of the microsomes was higher than that of the supernatant for short but not for long incubations, in agreement with observations on other systems. In this developing tissue the nuclei and especially the mitochondria appear to incorporate amino acid very rapidly. An insoluble fraction of the microsome pellet, which is presumably a liponucleoprotein complex, was found to possess, after 1 hour of incubation, a specific activity much greater than that of the purified microsomal particles or the supernatant fraction. Ninety-eight per cent of the leucine-C14 in the purified microsomal particles has been shown to possess bound amino groups, presumably in peptide linkages, by the DNP-end group method. These particles liberate but little peptide or protein of very high specific activity when they are destroyed by removal of Mg or by hydrolysis of RNA. Microsomal particles were fractionated into an RNA fraction and five protein fractions by means of density gradient centrifugation. By this method 95 per cent of the RNA can be separated from 90 per cent of the protein of the particle. Furthermore, the RNA fraction has been shown to contain very little protein of high specific activity. A particular protein fraction which contains the remaining 5 per cent of the RNA, possessed after 1 hour of incubation a specific activity 2 to 9 times higher than the protein of the other fractions.

On the Preparation of Bovine α-Thrombin

1978 ◽  
Vol 39 (01) ◽  
pp. 193-200 ◽  
Author(s):  
Erwin F Workman ◽  
Roger L Lundblad
Keyword(s):  
Immobilized Enzyme ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Low Molecular Weight ◽  
Reversible Process ◽  
Gel Beads ◽  
Tissue Thromboplastin ◽  
C 50 ◽  
Hydrolysis Of

SummaryAn improved method for the preparation of bovine α-thrombin is described. The procedure involves the activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2,200–2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strenght and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of α-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to “aged” thrombin. In addition, the catalytic properties of a-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Autoprothrombin C Activity from Prothrombin with Snake Venom or Trypsin

1962 ◽  
Vol 08 (03) ◽  
pp. 425-433 ◽  
Author(s):  
Ewa Marciniak ◽  
Edmond R Cole ◽  
Walter H Seegers
Keyword(s):  
Snake Venom ◽  
Thrombolytic Agent ◽  
Sodium Citrate ◽  
Specific Activity ◽  
High Specific Activity ◽  
Citrate Solution ◽  
Clotting Factors ◽  
Activation Products ◽  
Russell’S Viper ◽  
Autoprothrombin C

SummarySuitable conditions were found for the generation of autoprothrombin C from purified prothrombin with the use of Russell’s viper venom or trypsin. DEAE chromatographed prothrombin is structurally altered and has never been found to yield autoprothrombin C and also did not yield it when Russell’s viper venom or trypsin were used. Autoprothrombin C is derived from prothrombin with tissue extract thromboplastin, but not in large amounts with the intrinsic clotting factors. With the latter thrombin and autoprothrombin III are the chief activation products. Autoprothrombin III concentrates were prepared from serum and upon activation with 25% sodium citrate solution or with Russell’s viper venom large amounts of autoprothrombin C were obtained, and this was of high specific activity. Theoretically trypsin is not a thrombolytic agent, but on the contrary should lead to intravascular clotting.

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