Inactivation rates have been measured for clostridial glutamate dehydrogenase and several  engineered mutants at various DTNB concentrations. Analysis of rate constants  allowed determination of K<sub>d</sub> for each non-covalent enzyme-DTNB complex and the rate constant for reaction to form the inactive enzyme-thionitrobenzoate adduct. Both parameters are sensitive to the mutations F238S, P262S, the double mutation F238S/P262S, and D263K, all in the coenzyme binding site. Study of the effects of NAD<sup>+</sup>, NADH and NADPH at various concentrations in protecting against inactivation by 200 <svg style="vertical-align:-3.56265pt;width:9.5625px;" id="M1" height="12.175" version="1.1" viewBox="0 0 9.5625 12.175" width="9.5625" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg"><g transform="matrix(.017,-0,0,-.017,.062,7.675)"><path id="x1D707" d="M538 96q-38 -45 -84 -76.5t-71 -31.5q-44 0 -15 117q12 49 18 89h-2q-99 -128 -163 -178q-36 -28 -70 -28q-26 0 -44 38h-2q-16 -69 -16 -129q0 -43 11 -75.5t24 -42.5l1 -6q-7 -12 -24.5 -23t-37.5 -11q-40 0 -40 76q0 52 35 202l94 407l78 24l5 -5q-32 -122 -72 -310&#xA;q-7 -33 0 -55t25 -22q36 0 105.5 75t107.5 145l32 146l75 26h10q-50 -197 -78 -347q-7 -38 6 -38q7 0 31 17t47 39z" /></g></svg>M DTNB allowed determination of K<sub>d</sub> values for binding of these coenzymes to each protein, yielding surprising results. The mutations were originally devised to lessen discrimination against the disfavoured coenzyme NADP(H), and activity measurements showed this was achieved. However, the K<sub>d</sub> determinations indicated that, although K<sub>d</sub> values for NAD<sup>+</sup> and NADH were increased considerably, K<sub>d</sub> for NADPH was increased even more than for NADH, so that discrimination against binding of NADPH was not decreased. This apparent contradiction can only be explained if NADPH has a nonproductive binding mode that is not weakened by the mutations, and a catalytically productive mode that, though strengthened, is masked by the nonproductive binding. Awareness of the latter is important in planning further mutagenesis.

Joanna Griffin

Paul C. Engel

The –SH Protection Method for Determining Accurate K<sub><i>d</i></sub> Values for Enzyme-Coenzyme Complexes of NAD<sup><b>+</b></sup>-Dependent Glutamate Dehydrogenase and Engineered Mutants: Evidence for Nonproductive NADPH Complexes

On-site cellulase enzyme fermentation in a softwood-to-ethanol process, based on S<svg     style="vertical-align:-3.27605pt;width:19.1625px;" id="M1" height="15.5125" version="1.1" viewBox="0 0 19.1625 15.5125" width="19.1625"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,11.363)"><path id="x4F" d="M381 665q131 0 226.5 -93t95.5 -239q0 -158 -96 -253t-238 -95q-137 0 -231 95t-94 238q0 142 92.5 244.5t244.5 102.5zM359 629q-89 0 -151 -74.5t-62 -208.5q0 -141 69 -233t175 -92q90 0 150.5 74t60.5 211q0 152 -69 237.5t-173 85.5z" /></g>

			<g transform="matrix(.012,-0,0,-.012,12.763,15.45)"><path id="x32" d="M412 140l28 -9q0 -2 -35 -131h-373v23q112 112 161 170q59 70 92 127t33 115q0 63 -31 98t-86 35q-75 0 -137 -93l-22 20l57 81q55 59 135 59q69 0 118.5 -46.5t49.5 -122.5q0 -62 -29.5 -114t-102.5 -130l-141 -149h186q42 0 58.5 10.5t38.5 56.5z" /></g>

		
	
</svg>-catalysed steam pretreatment followed by simultaneous saccharification and fermentation, was investigated from a techno-economic aspect using Aspen Plus<svg     style="vertical-align:-0.1911pt;width:13.0375px;" id="M2" height="11.85" version="1.1" viewBox="0 0 13.0375 11.85" width="13.0375"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,11.55)"><path id="xA9" d="M531 248h16l-17 -76q-1 -10 -53 -21q-47 -10 -95 -10q-87 0 -139.5 51.5t-52.5 136.5q0 92 61 147q62 56 160 56q56 0 107 -22q13 -6 13 -17v-71h-17q-6 44 -33.5 67t-70.5 23q-65 0 -104 -48t-39 -127q0 -81 40.5 -128.5t107.5 -47.5q41 0 68.5 20.5t47.5 66.5zM722 330&#xA;q0 -144 -99 -245q-99 -99 -242 -99q-144 0 -244 100q-99 100 -99 244q0 146 100 246t248 100q140 0 237 -102q99 -100 99 -244zM667 334q0 123 -86 212q-85 88 -204 88q-115 0 -200 -91q-84 -90 -84 -212q0 -125 85 -214q86 -89 203 -89q118 0 202 90t84 216z" /></g>

		
	
</svg> and Aspen Icarus Process Evaluator<svg     style="vertical-align:-0.1911pt;width:13.0375px;" id="M3" height="11.85" version="1.1" viewBox="0 0 13.0375 11.85" width="13.0375"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,11.55)"><use xlink:href="#xA9"/></g>

		
	
</svg> softwares. The effect of varying the carbon source of enzyme fermentation, at constant protein and mycelium yields, was monitored through the whole process. Enzyme production step decreased the overall ethanol yield (270 L/dry tonne of raw material in the case of purchased enzymes) by 5&#x2013;16&#x2009;L/tonne. Capital cost was found to be the main cost contributor to enzyme fermentation, constituting to 60&#x2013;78&#x25; of the enzyme production cost, which was in the range of 0.42&#x2013;0.53&#x2009;SEK/L ethanol. The lowest minimum ethanol selling prices (4.71 and 4.82&#x2009;SEK/L) were obtained in those scenarios, where pretreated liquid fraction supplemented with molasses was used as carbon source. In some scenarios, on-site enzyme fermentation was found to be a feasible alternative.

Zsolt Barta

Krisztina Kovacs

Kati Reczey

Guido Zacchi

Process Design and Economics of On-Site Cellulase Production on Various Carbon Sources in a Softwood-Based Ethanol Plant

<i  >Mucor circinelloides</i> produced an extracellular polygalacturonase enzyme, the production of which was enhanced when various production parameters were optimized. Maximum polygalacturonase (PGase) activity was obtained in 48&#x2009;h at <svg     style="vertical-align:-0.20473pt;width:32.787498px;" id="M1" height="13.1875" version="1.1" viewBox="0 0 32.787498 13.1875" width="32.787498"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,12.875)"><path id="x33" d="M285 378v-2q65 -13 102 -54.5t37 -97.5q0 -57 -30.5 -104.5t-74 -75t-85.5 -42t-72 -14.5q-31 0 -59.5 11t-40.5 23q-19 18 -16 36q1 16 23 33q13 10 24 0q58 -51 124 -51q55 0 88 40t33 112q0 64 -39 96.5t-88 32.5q-29 0 -64 -11l-6 29q77 25 118 57.5t41 84.5&#xA;q0 45 -26.5 69.5t-68.5 24.5q-67 0 -120 -79l-20 20l43 63q51 56 127 56h1q66 0 107 -37t41 -95q0 -42 -31 -71q-22 -23 -68 -54z" /></g><g transform="matrix(.017,-0,0,-.017,8.222,12.875)"><path id="x30" d="M241 635q53 0 94 -28.5t63.5 -76t33.5 -102.5t11 -116q0 -58 -11 -112.5t-34 -103.5t-63.5 -78.5t-94.5 -29.5t-95 28t-64.5 75t-34.5 102.5t-11 118.5q0 58 11.5 112.5t34.5 103t64.5 78t95.5 29.5zM238 602q-32 0 -55.5 -25t-35.5 -68t-17.5 -91t-5.5 -105&#xA;q0 -76 10 -138.5t37 -107.5t69 -45q32 0 55.5 25t35.5 68.5t17.5 91.5t5.5 105t-5.5 105.5t-18 92t-36 68t-56.5 24.5z" /></g>

			<g transform="matrix(.012,-0,0,-.012,16.375,4.712)"><path id="x2218" d="M326 255q0 -56 -41 -96t-99 -40t-99 40t-41 96t41 96t99 40t99 -40t41 -96zM274 255q0 38 -25.5 65t-62.5 27t-62.5 -27t-25.5 -65t25.5 -65t62.5 -27t62.5 27t25.5 65z" /></g>

			<g transform="matrix(.017,-0,0,-.017,21.412,12.875)"><path id="x43" d="M614 175l29 -10q-33 -109 -57 -154q-121 -26 -184 -26q-90 0 -160.5 29t-112.5 77t-63.5 105.5t-21.5 119.5q0 157 108 253t277 96q36 0 71.5 -5t69 -13.5t36.5 -8.5q15 -102 20 -150l-29 -8q-20 79 -66.5 114t-128.5 35q-119 0 -187.5 -86t-68.5 -207&#xA;q0 -140 73.5 -227.5t188.5 -87.5q73 0 119.5 37.5t86.5 116.5z" /></g>

		
	
</svg> and pH 4.0 with pectin methyl ester (1&#x25; w/v) as carbon source and a combination of casein hydrolysate (0.1&#x25; w/v) and yeast extract (0.1&#x25; w/v) as nitrogen source. The enzyme was purified to homogeneity (13.3-fold) by Sephacryl S-100 gel-filtration chromatography. Its molecular weight was 66&#x2009;kDa on SDS-PAGE. The enzyme was found to have <svg     style="vertical-align:-3.3907pt;width:23.325001px;" id="M2" height="15.4" version="1.1" viewBox="0 0 23.325001 15.4" width="23.325001"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,11.112)"><path id="x1D43E" d="M764 650l-7 -26q-61 -10 -91.5 -22.5t-76.5 -48.5l-235 -184q104 -152 208 -276q31 -37 54.5 -48.5t68.5 -16.5l-7 -28h-156q-138 170 -213 284q-19 29 -33.5 35t-33.5 -7l-29 -173q-15 -75 -5.5 -90.5t74.5 -20.5l-5 -28h-260l7 28q58 5 74 21.5t30 89.5l70 378&#xA;q12 68 2 83t-72 22l6 28h257l-7 -28q-62 -7 -76 -21.5t-27 -83.5l-32 -172q29 11 78 46q112 84 217 179q18 16 23.5 25.5t-1 14.5t-26.5 8l-30 4l5 28h249z" /></g>

			<g transform="matrix(.012,-0,0,-.012,13.25,15.187)"><path id="x1D45A" d="M766 88q-40 -45 -83.5 -72.5t-63.5 -27.5q-39 0 -16 103l49 224q15 68 -10 68q-36 0 -112.5 -78.5t-105.5 -133.5q-27 -101 -38 -165q-35 -4 -77 -18l-6 6q42 153 70 288q12 55 10 78t-19 23q-38 0 -114 -80.5t-101 -131.5q-24 -68 -41 -165q-36 -3 -76 -18l-8 6&#xA;q56 202 87 347q7 33 -3 33q-8 0 -31.5 -17.5t-41.5 -35.5l-12 28q42 45 84 72t63 27q44 0 10 -124l-20 -75h2q92 121 193 178q36 21 64 21q62 0 28 -159l-8 -37h2q50 67 103.5 112t94.5 65q39 19 62 19q60 0 23 -156l-45 -189q-9 -39 2 -39q17 0 71 49z" /></g>

		
	
</svg> and <svg     style="vertical-align:-3.3907pt;width:35.924999px;" id="M3" height="15.4" version="1.1" viewBox="0 0 35.924999 15.4" width="35.924999"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,11.112)"><path id="x1D449" d="M730 650l-8 -28q-52 -4 -72 -18t-64 -77q-79 -113 -321 -539h-33l-119 541q-13 59 -29.5 73t-66.5 20l7 28h245l-8 -28l-28 -5q-33 -6 -40.5 -15.5t-0.5 -38.5l102 -450h2q191 320 246 430q21 42 15.5 56t-43.5 19l-31 4l7 28h240z" /></g>

			<g transform="matrix(.012,-0,0,-.012,12.675,15.187)"><path id="x6D" d="M797 0h-214v26q46 5 58 17.5t12 63.5v167q0 116 -88 116q-48 0 -112 -52q3 -18 3 -42v-195q0 -46 11 -58t53 -17v-26h-214v26q48 5 59.5 17t11.5 60v172q0 115 -88 115q-47 0 -109 -52v-235q0 -47 10 -59.5t48 -17.5v-26h-211v26q50 5 62.5 17t12.5 60v207q0 48 -9 59.5&#xA;t-52 18.5v23q79 14 139 41v-81q3 2 29 20.5t44 29.5q45 28 81 28t66 -22t46 -61q25 20 83 57q39 26 76 26q59 0 93 -40t34 -113v-193q0 -49 10.5 -60.5t54.5 -16.5v-26z" /></g><g transform="matrix(.012,-0,0,-.012,22.42,15.187)"><path id="x61" d="M433 39l-85 -51q-27 0 -47 20q-16 16 -24 44q-5 -3 -24 -16.5t-27 -18.5t-23 -13.5t-25 -12t-19 -3.5q-52 0 -86 36.5t-34 85.5q0 71 81 99q128 43 155 65v17q0 54 -23 83.5t-62 29.5q-28 0 -45 -19t-29 -66q-7 -23 -29 -23q-15 0 -29 13t-14 30t31 40q25 19 68.5 40.5&#xA;t81.5 29.5q49 0 82 -27q45 -39 45 -123v-185q0 -60 35 -60q18 0 36 11zM275 84v156q-12 -6 -49 -23t-41 -19q-31 -14 -46 -31t-15 -43q0 -35 22.5 -55.5t48.5 -20.5q19 0 42 10.5t38 25.5z" /></g><g transform="matrix(.012,-0,0,-.012,27.644,15.187)"><path id="x78" d="M474 0h-214v26q32 4 39 12t-5 28l-74 117q-39 -58 -64 -106q-16 -25 -10 -35t44 -16v-26h-186v26q46 6 64 17t48 49t85 120l-87 136q-23 36 -40 47t-58 16v26h199v-26q-31 -4 -36.5 -12.5t9.5 -29.5l59 -93q37 56 58 94q13 23 7 30t-40 11v26h183v-26q-47 -5 -65 -13.5&#xA;t-45 -39.5q-24 -29 -78 -110l97 -148q28 -43 46.5 -55.5t63.5 -18.5v-26z" /></g>

		
	
</svg> values of 2.2&#x2009;mM and 4.81&#x2009;IU/ml at 0.1&#x25; to 0.5&#x25; (w/v) concentration of the substrate. The  addition of phenolic acids (0.05&#x2009;mM), metal ions such as Mn<sup  >+2</sup>, Co<sup  >+2</sup>, Mg<sup  >+2</sup>, <svg     style="vertical-align:-0.16379pt;width:29.3375px;" id="M4" height="16.049999" version="1.1" viewBox="0 0 29.3375 16.049999" width="29.3375"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,15.775)"><path id="x46" d="M493 503l-29 -4q-13 56 -28 80q-13 22 -35.5 29.5t-78.5 7.5h-76q-22 0 -27.5 -5t-5.5 -27v-238h103q66 0 82.5 12t25.5 62h29v-193h-29q-9 54 -25.5 67t-82.5 13h-103v-181q0 -64 15 -78.5t84 -19.5v-28h-272v28q61 5 74.5 19.5t13.5 78.5v399q0 64 -13 78t-72 19v28&#xA;h440q1 -40 10 -147z" /></g><g transform="matrix(.017,-0,0,-.017,8.732,15.775)"><path id="x65" d="M380 106l15 -22q-42 -52 -87 -74t-76 -22q-89 0 -142 62t-53 151q0 103 66 175q67 73 146 73h1q61 0 105.5 -46.5t44.5 -105.5q0 -16 -4.5 -22.5t-18.5 -9.5q-54 -10 -262 -17q1 -95 46 -143.5t104 -48.5q61 0 115 50zM225 412q-35 0 -64.5 -32.5t-40.5 -90.5q83 0 170 3&#xA;q14 0 19 4.5t5 15.5q1 41 -23.5 70.5t-65.5 29.5z" /></g>

			<g transform="matrix(.012,-0,0,-.012,15.95,7.613)"><path id="x2B" d="M535 230h-212v-233h-58v233h-213v50h213v210h58v-210h212v-50z" /></g><g transform="matrix(.012,-0,0,-.012,22.935,7.613)"><use xlink:href="#x33"/></g>

		
	
</svg>, <svg     style="vertical-align:-0.0pt;width:29.5375px;" id="M5" height="15.8375" version="1.1" viewBox="0 0 29.5375 15.8375" width="29.5375"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,15.775)"><path id="x41" d="M673 0h-245v28q47 6 58 16t1 39l-55 154h-213q-31 -87 -36 -129q-16 -47 -5 -61t68 -19v-28h-226v28q50 7 68.5 23.5t43.5 80.5l214 524l24 9l193 -536q23 -63 41.5 -79.5t68.5 -21.5v-28zM418 280l-81 255h-2q-74 -197 -101 -255h184z" /></g><g transform="matrix(.017,-0,0,-.017,11.843,15.775)"><path id="x6C" d="M238 0h-223v26q48 5 60 17t12 61v473q0 50 -10.5 60.5t-58.5 15.5v24q73 8 148 35v-608q0 -49 11.5 -61t60.5 -17v-26z" /></g>

			<g transform="matrix(.012,-0,0,-.012,16.162,7.613)"><use xlink:href="#x2B"/></g><g transform="matrix(.012,-0,0,-.012,23.147,7.613)"><use xlink:href="#x33"/></g>

		
	
</svg>, Hg<sup  >+2</sup>, and Cu<sup  >+2</sup>, and thiols had inhibitory effect on the enzyme. The enzyme showed maximum activity in the presence of polygalacturonic acid (0.1&#x25; w/v) at pH 5.5 and <svg     style="vertical-align:-0.20473pt;width:32.787498px;" id="M6" height="13.1875" version="1.1" viewBox="0 0 32.787498 13.1875" width="32.787498"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,12.875)"><path id="x34" d="M456 178h-96v-72q0 -51 12.5 -62.5t72.5 -16.5v-27h-256v27q65 5 78 17t13 62v72h-260v28q182 271 300 426h40v-407h96v-47zM280 225v295h-2q-107 -148 -196 -295h198z" /></g><g transform="matrix(.017,-0,0,-.017,8.222,12.875)"><path id="x32" d="M412 140l28 -9q0 -2 -35 -131h-373v23q112 112 161 170q59 70 92 127t33 115q0 63 -31 98t-86 35q-75 0 -137 -93l-22 20l57 81q55 59 135 59q69 0 118.5 -46.5t49.5 -122.5q0 -62 -29.5 -114t-102.5 -130l-141 -149h186q42 0 58.5 10.5t38.5 56.5z" /></g>

			<g transform="matrix(.012,-0,0,-.012,16.375,4.712)"><use xlink:href="#x2218"/></g>

			<g transform="matrix(.017,-0,0,-.017,21.412,12.875)"><use xlink:href="#x43"/></g>

		
	
</svg>.

Akhilesh Thakur

Roma Pahwa

Smarika Singh

Reena Gupta

Production, Purification, and Characterization of Polygalacturonase from <i>Mucor circinelloides</i> ITCC 6025

Amylases are one of the most important industrial enzymes produced worldwide, with their major application being in ethanol manufacturing. This work investigated the production of amylases by solid-state fermentation of babassu cake, using the filamentous fungus <i  >Aspergillus awamori</i> IOC-3914. Lab-scale experiments were carried out to generate input data for simulations of an industrial plant for amylase production. Additionally to the target enzymes, other hydrolases (cellulases, xylanases, and proteases) were also produced, enriching the final product. The most suitable fermentation time was 144 hours, when exoamylase and endoamylase activities of 40.5 and 42.7&#x2009;U&#x2009;<svg     style="vertical-align:-3.50803pt;width:21.4px;" id="M1" height="20.2125" version="1.1" viewBox="0 0 21.4 20.2125" width="21.4"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
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			<g transform="matrix(.012,-0,0,-.012,8.012,7.613)"><path id="x2212" d="M535 230h-483v50h483v-50z" /></g><g transform="matrix(.012,-0,0,-.012,14.997,7.613)"><path id="x31" d="M384 0h-275v27q67 5 81.5 18.5t14.5 68.5v385q0 38 -7.5 47.5t-40.5 10.5l-48 2v24q85 15 178 52v-521q0 -55 14.5 -68.5t82.5 -18.5v-27z" /></g>

		
	
</svg> were achieved, respectively. A first evaluation showed a large impact of the inoculum propagation medium on production costs. Therefore, five propagation media were compared, and PDA medium presented the best cost-benefit ratio. The credits obtained from sales of fermented cake as a coproduct enabled a significant decrease in the production cost of the enzyme product, down to 10.40&#x2009;USD&#x2009;<svg     style="vertical-align:-3.50803pt;width:29.825001px;" id="M2" height="20.2125" version="1.1" viewBox="0 0 29.825001 20.2125" width="29.825001"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,15.775)"><path id="x6B" d="M513 0l-137 -2q-16 18 -164 198q-19 22 -31 22q-7 0 -16 -3v-111q0 -49 11.5 -61t60.5 -17v-26h-223v26q50 4 61.5 17t11.5 61v472q0 50 -10 61.5t-56 15.5v24q57 6 144 35v-471q37 7 63 35q31 31 71 88q16 23 10.5 31.5t-40.5 12.5v26l204 9v-25q-51 -8 -75 -19.5&#xA;t-54 -39.5q-31 -31 -86 -95q13 -21 70 -87.5t85 -95.5q48 -52 100 -55v-26z" /></g><g transform="matrix(.017,-0,0,-.017,8.494,15.775)"><use xlink:href="#x67"/></g>

			<g transform="matrix(.012,-0,0,-.012,16.45,7.613)"><use xlink:href="#x2212"/></g><g transform="matrix(.012,-0,0,-.012,23.435,7.613)"><use xlink:href="#x31"/></g>

		
	
</svg>.

Aline Machado de Castro

Daniele Fernandes Carvalho

Denise Maria Guimarães Freire

Leda dos Reis Castilho

Economic Analysis of the Production of Amylases and Other Hydrolases by <i>Aspergillus awamori</i> in Solid-State Fermentation of Babassu Cake

The gene encoding <small class="sc">D</small>-phenylserine dehydrogenase from <i  >Pseudomonas syringae</i> NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that <i  >orf3</i> encoded <small class="sc">L</small>-phenylserine dehydrogenase. Hence, <i  >orf3</i> was cloned and overexpressed in <i  >Escherichia coli</i> cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed <small class="sc">L</small>-phenylserine dehydrogenase activity. The enzymological properties and primary structure of <small class="sc">L</small>-phenylserine dehydrogenase (ORF3) were quite different from those of <small class="sc">D</small>-phenylserine dehydrogenase previously reported. <small class="sc">L</small>-Phenylserine dehydrogenase catalyzed the <svg     style="vertical-align:-0.12283pt;width:43.962502px;" id="M1" height="14.2625" version="1.1" viewBox="0 0 43.962502 14.2625" width="43.962502"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
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</svg>-dependent oxidation of the <svg     style="vertical-align:-3.56265pt;width:10px;" id="M2" height="16.6625" version="1.1" viewBox="0 0 10 16.6625" width="10"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
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</svg>-hydroxyl group of <small class="sc">L</small>-<svg     style="vertical-align:-3.56265pt;width:10px;" id="M3" height="16.6625" version="1.1" viewBox="0 0 10 16.6625" width="10"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
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</svg>-phenylserine. <small class="sc">L</small>-Phenylserine and <small class="sc">L</small>-<i  >threo</i>-(2-thienyl)serine were good substrates for <small class="sc">L</small>-phenylserine dehydrogenase. The genes encoding <small class="sc">L</small>-phenylserine dehydrogenase and <small class="sc">D</small>-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and <svg     style="vertical-align:-3.27605pt;width:30.575001px;" id="M4" height="15.5125" version="1.1" viewBox="0 0 30.575001 15.5125" width="30.575001"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
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</svg>.

Sakuko Ueshima

Hisashi Muramatsu

Takanori Nakajima

Hiroaki Yamamoto

Shin-ichiro Kato

Haruo Misono

Shinji Nagata

Identification, Cloning, and Characterization of <small class="sc">l</small>-Phenylserine Dehydrogenase from <i>Pseudomonas syringae</i> NK-15

The effects of exposure to hyperbaric oxygen on the oxidative capacity of the skeletal muscles in mice at different ages were investigated. We exposed 5-, 34-, 55-, and 88-week-old mice to 36% oxygen at 950 mmHg for 6 hours per day for 2 weeks. The activities of succinate dehydrogenase (SDH), which is a mitochondrial marker enzyme, of the tibialis anterior muscle in hyperbaric mice were compared with those in age-matched mice under normobaric conditions (21% oxygen at 760 mmHg). Furthermore, the SDH activities of type IIA and type IIB fibers in the muscle were determined using quantitative histochemical analysis. The SDH activity of the muscle in normobaric mice decreased with age. Similar results were observed in both type IIA and type IIB fibers in the muscle. The decrease in the SDH activity of the muscle was reduced in hyperbaric mice at 57 and 90 weeks. The decreased SDH activities of type IIA and type IIB fibers were reduced in hyperbaric mice at 90 weeks and at 57 and 90 weeks, respectively. We conclude that exposure to hyperbaric oxygen used in this study reduces the age-related decrease in the oxidative capacity of skeletal muscles.

Takahiro Nishizaka

Fumiko Nagatomo

Hidemi Fujino

Tomoko Nomura

Tomohiko Sano

Kazuhiko Higuchi

Isao Takeda

Akihiko Ishihara

Hyperbaric Oxygen Exposure Reduces Age-Related Decrease in Oxidative Capacity of the Tibialis Anterior Muscle in Mice

This study was designed to investigate the effects of molluscicidal components of <i>Myristica fragrans</i> Houtt. (Myristicaceae) on certain enzymes in the nervous tissue of freshwater snail <i>Lymnaea acuminata</i> Lamarck (Lymnaeidae). <i>In vivo</i> and <i>in vitro</i> treatments of trimyristin and myristicin (active molluscicidal components of <i>Myristica fragrans</i> Houtt.) significantly inhibited the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activities in the nervous tissue of <i>Lymnaea acuminata</i>. The inhibition kinetics of these enzymes indicates that both the trimyristin and myristicin caused competitive noncompetitive inhibition of AChE. Trimyristin caused uncompetitive and competitive/noncompetitive inhibitions of ACP and ALP, respectively whereas the myristicin caused competitive and uncompetitive inhibition of ACP and ALP, respectively. Thus results from the present study suggest that inhibition of AChE, ACP, and ALP by trimyristin and myristicin in the snail <i>Lymnaea acuminata</i> may be the cause of the molluscicidal activity of <i>Myristica fragrans</i>.

Preetee Jaiswal

Pradeep Kumar

V. K. Singh

D. K. Singh

Enzyme Inhibition by Molluscicidal Components of <i>Myristica fragrans</i> Houtt. in the Nervous Tissue of Snail <i>Lymnaea acuminata</i>

L-asparaginase was extracted from <i  >Erwinia carotovora</i> and purified by ammonium sulfate fractionation (60&#x2013;70&#x25;), Sephadex G-100, CM cellulose, and DEAE sephadex chromatography. The apparent Mr of enzyme under nondenaturing and denaturing conditions was 150&#x2009;kDa and <svg style="vertical-align:-1.09097pt;width:52.9375px;" id="M1" height="12.2375" version="1.1" viewBox="0 0 52.9375 12.2375" width="52.9375"  xmlns="http://www.w3.org/2000/svg">
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</svg>&#x2009;kDa, respectively. L-asparaginase activity was studied in presence of thiols, namely, L-cystine (Cys), L-methionine (Met), N-acetyl cysteine (NAC), and reduced glutathione (GSH). Kinetic parameters in presence of thiols (10&#x2013;400 <svg style="vertical-align:-2.29482pt;width:9.6374998px;" id="M2" height="9.6750002" version="1.1" viewBox="0 0 9.6374998 9.6750002" width="9.6374998"  xmlns="http://www.w3.org/2000/svg">
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</svg>M) showed an increase in <svg style="vertical-align:-3.22281pt;width:30.875px;" id="M3" height="14.4875" version="1.1" viewBox="0 0 30.875 14.4875" width="30.875"  xmlns="http://www.w3.org/2000/svg">
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</svg> values (2000, 2223, 2380, 2500, and control 1666.7&#x2009;<svg style="vertical-align:-2.29482pt;width:9.6374998px;" id="M4" height="9.6750002" version="1.1" viewBox="0 0 9.6374998 9.6750002" width="9.6374998"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,9.675)">
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</svg>) and a decrease in <svg style="vertical-align:-3.20526pt;width:19.799999px;" id="M7" height="14.4625" version="1.1" viewBox="0 0 19.799999 14.4625" width="19.799999"  xmlns="http://www.w3.org/2000/svg">
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</svg> values (0.086, 0.076, 0.062, 0.055 and control 0.098&#x2009;mM) indicating nonessential mode of activation. <svg style="vertical-align:-3.13504pt;width:19.924999px;" id="M8" height="14.375" version="1.1" viewBox="0 0 19.924999 14.375" width="19.924999"  xmlns="http://www.w3.org/2000/svg">
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</svg> values displayed propensity to bind thiols. A decrease in <svg style="vertical-align:-3.22281pt;width:54.887501px;" id="M9" height="14.7125" version="1.1" viewBox="0 0 54.887501 14.7125" width="54.887501"  xmlns="http://www.w3.org/2000/svg">
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				<tspan style="font-size: 8.75px; " x="6.8090558" y="0">a</tspan>
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</svg> ratio in concentration plots showed inverse relationship between free thiol groups (NAC and GSH) and bound thiol group (Cys and Met). Enzyme activity was enhanced in presence of thiol protecting reagents like dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and GSH, but inhibited by p-chloromercurybenzoate (PCMB) and iodoacetamide (IA).

Suchita C. Warangkar

Chandrahas N. Khobragade

Purification, Characterization, and Effect of Thiol Compounds on Activity of the <i>Erwinia carotovora</i> L-Asparaginase

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Enzyme Research publishes original research articles as well as review articles that advance our understanding of enzymes and the way in which they work, both in vivo and in vitro.

Enzyme Research