Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

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Characterization of a Mannose-6-Phosphate Isomerase fromThermus thermophilusand Increased l-Ribose Production by Its R142N Mutant

Applied and Environmental Microbiology ◽

10.1128/aem.01793-10 ◽

2010 ◽

Vol 77 (3) ◽

pp. 762-767 ◽

Cited By ~ 17

Author(s):

Soo-Jin Yeom ◽

Eun-Sun Seo ◽

Bi-Na Kim ◽

Yeong-Su Kim ◽

Deok-Kun Oh

Keyword(s):

Specific Activity ◽

Thermus Thermophilus ◽

Catalytic Efficiency ◽

Maximal Activity ◽

Wild Type ◽

Active Site Residues ◽

Geobacillus Thermodenitrificans ◽

Wild Type Enzyme ◽

Mannose 6 Phosphate

ABSTRACTAn uncharacterized gene fromThermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed inEscherichia coli. The maximal activity of the recombinant enzyme forl-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion ofl-ribulose tol-ribose, a potential starting material for manyl-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase inl-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. Thekcat/Kmof the R142N mutant was 3.8-fold higher than that ofGeobacillus thermodenitrificansmannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reportedkcat/Km. The R142N mutant enzyme produced 213 g/literl-ribose from 300 g/literl-ribulose for 2 h, with a volumetric productivity of 107 g liter−1h−1, which was 1.5-fold higher than that of the wild-type enzyme.

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Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.00457-13 ◽

2013 ◽

Vol 79 (13) ◽

pp. 4072-4077 ◽

Cited By ~ 57

Author(s):

Xuguo Duan ◽

Jian Chen ◽

Jing Wu

Keyword(s):

Catalytic Efficiency ◽

Kinetic Studies ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Ph Optimum ◽

Widespread Application ◽

Content Type ◽

Major Factors

ABSTRACTPullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase fromBacillus deramificanswere selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that theKmvalues for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and theKcat/Kmvalues increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

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Replacement of Tyrosine 181 by Phenylalanine in Gentisate 1,2-Dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 Enhances Catalytic Activities

Journal of Bacteriology ◽

10.1128/jb.187.21.7543-7545.2005 ◽

2005 ◽

Vol 187 (21) ◽

pp. 7543-7545 ◽

Cited By ~ 3

Author(s):

Chew Ling Tan ◽

Chew Chieng Yeo ◽

Hoon Eng Khoo ◽

Chit Laa Poh

Keyword(s):

Catalytic Efficiency ◽

Relative Activity ◽

Site Directed Mutagenesis ◽

Mutant Enzyme ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Specific Mutation ◽

Catalytic Activities ◽

Pseudomonas Alcaligenes

ABSTRACT xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k cat/Km ) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.

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Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation

Biochemical Journal ◽

10.1042/bj3010275 ◽

1994 ◽

Vol 301 (1) ◽

pp. 275-281 ◽

Cited By ~ 35

Author(s):

H M Chen ◽

C Ford ◽

P J Reilly

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Membrane ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Aspergillus Awamori ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Recognition Sites ◽

Membrane Components

Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.

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Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase

Biochemical Journal ◽

10.1042/bj3260047 ◽

1997 ◽

Vol 326 (1) ◽

pp. 47-51 ◽

Cited By ~ 16

Author(s):

Shoshana KEYNAN ◽

Nigel M. HOOPER ◽

Anthony J. TURNER

Keyword(s):

Specific Activity ◽

Mammalian Species ◽

Competitive Inhibitor ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Renal Metabolism ◽

Leukotriene D4 ◽

Histidine Residues

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

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Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

Journal of Bacteriology ◽

10.1128/jb.00259-06 ◽

2006 ◽

Vol 188 (17) ◽

pp. 6179-6183 ◽

Cited By ~ 16

Author(s):

Jung-Kul Lee ◽

Ee-Lui Ang ◽

Huimin Zhao

Keyword(s):

Substrate Specificity ◽

Mutational Analysis ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Saturation Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Enzyme Substrate ◽

Molecular Determinants

ABSTRACT Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.

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Improvement in the Thermostability of d-Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.05566-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7316-7320 ◽

Cited By ~ 31

Author(s):

Jin-Geun Choi ◽

Yo-Han Ju ◽

Soo-Jin Yeom ◽

Deok-Kun Oh

Keyword(s):

Enzyme Activity ◽

Agrobacterium Tumefaciens ◽

Operation Time ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Conversion Yield ◽

Content Type ◽

Aromatic Stacking

ABSTRACTThe S213C, I33L, and I33L S213C variants ofd-psicose 3-epimerase fromAgrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer ofd-psicose.

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Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.71.9.5290-5296.2005 ◽

2005 ◽

Vol 71 (9) ◽

pp. 5290-5296 ◽

Cited By ~ 11

Author(s):

Wei Zhang ◽

Yuan Liu ◽

Huabao Zheng ◽

Sheng Yang ◽

Weihong Jiang

Keyword(s):

Specific Activity ◽

Catalytic Efficiency ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Wild Type ◽

Catalytic Function ◽

Alkaline Shift ◽

Key Residues

ABSTRACT In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k cat/Km ) compared to wild-type CA130. Their Km values were decreased by ca. 50%, and the k cat values increased to 14.4 and 16.9 s−1, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.

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Active-site serine mutants of the Streptomyces albus G β-lactamase

Biochemical Journal ◽

10.1042/bj2770647 ◽

1991 ◽

Vol 277 (3) ◽

pp. 647-652 ◽

Cited By ~ 10

Author(s):

F Jacob ◽

B Joris ◽

J M Frère

Keyword(s):

Active Site ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Beta Lactamase ◽

Wild Type ◽

Serine Residue ◽

Wild Type Enzyme ◽

Ph Values ◽

Streptomyces Albus ◽

Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj2790035 ◽

1991 ◽

Vol 279 (1) ◽

pp. 35-41 ◽

Cited By ~ 86

Author(s):

R Chambert ◽

M F Petit-Glatron

Keyword(s):

Bacillus Subtilis ◽

Intermediate Formation ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Chain Elongation ◽

Directed Mutagenesis ◽

Water Mixture ◽

Wild Type ◽

Wild Type Enzyme ◽

Sucrose Hydrolysis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

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