Isolation and properties of a constitutive D-xylulokinase from a novel thermophilic Saccharococcus caldoxylosilyticus DSM 12041 (ATCC 700356) Shafiq Ahmad 1, *, Robert K. Scopes School of Biochemistry, La Trobe University, Bundoora, Melbourne Victoria 3083, Australia Received 16 July 2001; received in revised form 10 October 2001; accepted 22 October 2001 Abstract D-Xylulokinase (ATP: D-xylulose 5-phosphotransferase EC 2.7.1.17) was purified from a newly isolated thermophilic Saccharococcus caldoxylosilyticus in which the enzyme is constitutively expressed. The purified enzyme had a specific activity of 60 U/mg at 25°C, and 185 U/mg in its optimum temperature range of between 65 and 75°C. Its K m for xylulose was 0.09 mM at 25°C, and for MgATP 0.16 mM. The molecular mass of the monomer was estimated to be 54 kDa, the holoenzyme comprising two subunits. Stability studies showed that the enzyme was stable up to 65°C, but denatured rapidly at 75°C in Tris buffer. However, it was stabilised by xylulose, which accounts for its high optimum temperature for activity. N-terminal amino acid analysis produced the sequence DHVIGVDLGTSAVKALLVD . . . which has 65% identity with two other Bacillus xylulokinases as deduced from their gene sequences, and somewhat less identity with other known xylulokinase sequences. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Xylulokinase; Saccharococcus; Enzyme purification; Thermophile; Xylose metabolism 1. Introduction There is much interest in utilising renewable plant bio- mass for production of fuels and chemical feedstocks. Lignocellulosic materials include substantial amounts of xylans which on hydrolysis either enzymically or chemi- cally, yield mainly xylose. The biodegradation of xylans has been much studied in recent years, with particular emphasis on the xylanases, which have proved useful in some bio- technological process. Several xylanases from thermophilic bacteria have been isolated and introduced into paper pulp production because of their superior thermostability to xy- lanases from mesophiles [1,2]. The use of lignocellulosic biomass, for biofuel production is being intensively inves- tigated [3– 6]. Xylans can be readily hydrolysed and the liberated xylose can be fermented rapidly by many bacteria [7]. The first step in bacterial D-xylose metabolism is isom- erisation to D-xylulose, which is then phosphorylated and further metabolised by pentose phosphate pathway en- zymes. Xylose isomerases are used commercially on a large scale for the conversion of glucose to high-fructose syrup. Thus, both xylanases and xylose isomerases have been isolated from many bacteria, including thermophiles, and extensively characterised. But the next enzyme in the xylose utilisation pathway, xylulokinase, has not to date been de- scribed from a thermophile. D-xylulokinase has been isolated from some mesophilic bacteria, from yeast [8] and bovine liver [9]. However, despite its importance in xylan degradation, there are few detailed studies of this enzyme; the only ones from bacteria are those of Aerobacter (Klebsiella) aerogenes [7,10], and a much earlier description of the enzyme from Lactobacillus pentosus [11]. Complete gene sequences have been reported for about ten species’ xylulokinases to date, none of which is a thermophile. We describe here the isolation and prop- erties of D-xylulokinase from a newly isolated species of a thermophile Saccharococcus named caldoxylosilyticus. The strain Saccharococcus caldoxylosilyticus S1812 = ATCC 700356 = DSM 12041 has been deposited at culture col- lections Deutsche Sammlung von Mikroorganismen und * Corresponding author. Tel.: +61-3-9545-2272; fax: +61-3-9545- 2446. E-mail address: Shafiq.Ahmad@molsci.csiro.au (S. Ahmad). 1 Current address: CSIRO Molecular Science, Private Bag 10, Clayton South, Melbourne Victoria 3169, Australia. www.elsevier.com/locate/enzmictec Enzyme and Microbial Technology 30 (2002) 627– 632 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0141-0229(01)00518-X