Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis.

AIMS: The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus. METHODS AND RESULTS: The 750 bp xylanase gene was amplified and subcloned into the unique NheI restriction enzyme site of pMG36e and subsequently transformed into competent Escherichia coli XLI-blue MRF cells and Lactococcus lactis cells. Bacterial culture containing pMG36e-Xy has an enzyme activity of 390 microg xylose ml(-1) culture 30 min(-1), respectively, when compared with 40 microg xylose ml(-1) culture 30 min(-1) for the negative control (plasmidless strain). CONCLUSIONS: The thermostable xylanase gene was successfully expressed in both E. coli and L. lactis. The activity of xylanase can be easily detected by the formation of visible clearing zones around the transformed colonies on Remazol Brilliant Blue-Xylan (RBB-Xylan) agar media. However, there were some significant differences in the optimum growth temperature and plasmid stability in the new clones. SIGNIFICANCE AND IMPACT OF THE STUDY: The constructed reporter vector has the potential to be used as a reporter system for Lactococcus as well as E. coli, and it is an addition to the pool of lactococcal vector systems.

Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli.

A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).

beta-hydroxy-beta-methylglutaryl coenzyme A hydrolase of rat liver.

Beta-Hydroxy-beta-methylglutaryl coenzyme A hydrolase, or deacylase, (EC 3.1.2.5) is important, at least potentially, in the regulation of mammalian cholesterol synthesis. This is so for two reasons, both related to the enzyme generally regarded as rate-limiting for cholesterogenesis, namely beta-hydroxy-beta-methylglutaryl CoA reductase: (i) the hydrolase competes for the same substrate as the reductase and (ii) its end product, hydroxymethylglutamic acid, is a known inhibitor of the reductase. Consequently we have examined some of the properties of the hydrolase, as found in rat liver, after first developing a simple isotopic technique for its assay. Beta-Hydroxy-beta-methylglutaryl CoA hydrolase is both soluble and microsomal. The microsomal enzyme is inactivated by pre-incubation at 37 degree C, but not a 4 degree C, has an apparent pH optimum of approximately 7.6, and has Km and V values of 270 (microM) and 33.3 (nmol HMG/10 per mg protein), respectively, at 37 degree C. For the cytosolic enzyme the corresponding Km and V values are 830 and 111.1. From our observations it seems unlikely that beta-hydroxy-beta-methylglutaryl CoA hydrolase plays a significant role in the regulation of hepatic cholesterol synthesis since, in contrast to microsomal beta-hydroxy-beta-methylglutaryl coenzyme A reductase, we could find for the microsomal hydrolase no evidence of a diurnal rhythm of activity, no inhibition of activity by short-term cholesterol feeding and no evidence from Arrhenius-plot data for any membrane-mediated control of enzyme activity. Thus, the significance of the enzyme in mammalian systems remains unknown.

Membrane-mediated control of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase.

Previously we [Sabine & James (1976) Life Sci. 18, 1185--1192] proposed that 'the activity of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase is critically regulated by the fluidity of its supporting microsomal membrane'. In the present work we examined further this concept of membrane-mediated control, with respect to the specific hypothesis that such control might function as a common mechanism both for the co-ordinated regulation of other enzymes affected by cholesterol feeding and also for the subcellular integration of the several physiological factors known to influence this enzyme's activity. Contrary to earlier expectations, this hypothesis now appears not to hold. We report here that, under those conditions of short-term cholesterol feeding that affected the reductase, a variety of other microsomal enzymes did not display membrane-function interactions, i.e. neither enzymes involved in cholesterol metabolism and also affected by cholesterol feeding (cholesterol 7 alpha-hydroxylase), nor those involved in cholesterol metabolism and not affected by cholesterol feeding (hydroxymethylglutaryl-CoA hydrolase, acyl-CoA:cholesterol acyltransferase), nor those not directly involved in cholesterol metabolism at all (glucose 6-phosphatase). Furthermore, we observed no evidence for the operation of membrane-mediated control of the reductase in other situations known to influence its activity, i.e. starvation, diurnal rhythm, the very early stages of cholesterol feeding and various manipulations in vitro.