Elsevier

Process Biochemistry

Volume 48, Issue 1, January 2013, Pages 109-117
Process Biochemistry

Discovery and characterization of a thermostable d-lactate dehydrogenase from Lactobacillus jensenii through genome mining

https://doi.org/10.1016/j.procbio.2012.11.013Get rights and content

Abstract

The demand on thermostable d-lactate dehydrogenases (d-LDH) has been increased for d-lactic acid production but thermostable d-DLHs with industrially applicable activity were not much explored. To identify a thermostable d-LDH, three d-LDHs from different Lactobacillus jensenii strains were screened by genome mining and then expressed in Escherichia coli. One of the three d-LDHs (d-LDH3) exhibited higher optimal reaction temperature (50 °C) than the others. The T5010 value of this thermostable d-LDH3 was 48.3 °C, much higher than the T5010 values of the others (42.7 and 42.9 °C) and that of a commercial d-lactate dehydrogenase (41.2 °C). The Tm values were 48.6, 45.7 and 55.7 °C for the three d-LDHs, respectively. In addition, kinetic parameter (kcat/Km) of d-LDH3 for pyruvate reduction was estimated to be almost 150 times higher than that for lactate oxidation at pH 8.0 and 25 °C, implying that d-lactate production from pyruvate is highly favored. These superior thermal and kinetic features would make the d-LDH3 characterized in this study a good candidate for the microbial production of d-lactate at high temperature from glucose if it is genetically introduced to lactate producing microbial.

Highlights

► Three kinds of d-lactate dehydrogenase from genome of Lactobacillus jensenii were expressed in E. coli.d-LDH3 exhibited higher optimal temperature up to 50 °C compared with those of other d-LDHs. ► The catalytic efficiency (kcat/Km) of d-LDH3 for pyruvate conversion was estimated to be 200 times higher than that for lactate conversion at pH 8.0 and 25 °C. ► The d-LDH3 can be a good candidate biocatalyst for the d-lactate production at high temperature.

Introduction

Lactic acid, a monomer required for the production of polylactic acid (PLA), is the major end product of carbohydrate fermentation by industrially important homofermentative lactic acid bacteria. Two isomers of lactic acid, the dextrorotatory (d-) and levorotatory (l-) isomers, can be produced by lactate dehydrogenases (LDHs). Optically pure isomers can be produced as separate products using chiral-specific D- or L-lactate dehydrogenase (d- or l-LDH) enzymes [1]. d-LDHs catalyze the NAD-dependent conversion of pyruvate to d-lactic acid and the reverse reaction [2].

l-LDHs have been thoroughly studied because l-lactic acid is widely used in food, cosmetics and medicine [3], [4]. In contrast, there are few applications of d-lactic acid [5], [6], and detailed research on d-LDHs has been comparatively neglected [7], [8]. Increasing interest in stereocomplex PLA, comprising PDLA and PLLA, which has better properties than racemic PLA – such as increased melting point and impact strength – has boosted the production of optically pure d-lactic acid, which is required for the preparation of PDLA [9], [10], [11]. l-Lactic acid can be produced commercially by microbial fermentation at high yields and titers at temperatures between 30 and 40 °C [12], [13]. However, despite the fact that several lactic acid bacteria, such as Lactobacillus delbrueckii and Lactobacillus coryniformis, produce d-lactic acid rather than l-lactic acid [14], [15], [16], the productivity of d-lactic acid fermentation using Lactobacillus was relatively lower than that of l-lactic acid fermentation at 40 °C incubation temperature, which may imply that thermostabilty of d-LDHs can be weaker than that of l-LDHs. Compared with thermostable l-LDHs [17], thermostable d-LDHs have not been well characterized at genetic level, and d-LDHs have lower thermostabilities than the l-LDHs in the same hosts [18], [19]. Hyper-thermostable d-LDHs have been found in thermophiles such as Sulfolobus tokodaii, but these enzymes cannot be used in the production of d-lactic acid because thermophilic enzymes are not highly active at normal culture temperatures (∼40 °C) [20]. Wang et al. successfully produced d-lactate by microbial fermentation at 50 °C using a thermostable d-LDH [12]. However, this enzyme had evolved from a glycerol dehydrogenase, and it had dual activities toward glycerol and d-lactate. Therefore, the discovery or engineering of thermostable d-LDHs, which should be active at culture temperatures above 40 °C, is necessary for the economical production of d-lactic acid.

This work attempted to search for thermostable d-LDHs that could convert pyruvate into d-lactic acid. Even though the d-LDHs in supernatant of Lactobacillus jensenii culture broth were reported active at up to 50 °C [21], however there has been no available information for these d-LDHs including gene and protein sequence. We have tried the functional expression of genes encoding three L. jensenii d-LDHs in E. coli to find and characterize thermostable d-LDHs up to 50 °C.

Section snippets

Phylogenetic analysis

Genes encoding d-LDHs were identified in these all Lactobacillus types which are L. jensenii 1153 (ABWG00000000), L. jensenii JV-V16 (ACGQ00000000), L. jensenii 27-2-CHN (ACOF00000000), L. jensenii 269-3 (ACOY00000000), L. jensenii SJ-7A-US (ACQD00000000), L. jensenii 115-3-CHN (ACQN00000000) and L. jensenii 208-1 (ADEX00000000) and their relatives in the GenBank database. The retrieved sequences were aligned, and the phylogenetic tree for d-LDH was generated using the maximum-likelihood

Extraction of the d-LDH genes from the genome of L. jensenii and their amino acid compositions

The d-LDH genes of L. jensenii were clustered in two monophyletic groups in the phylogenetic tree (Fig. 1). Cluster I formed a monophyletic group with genes from L. gasseri, L. johnsonii and L. acidophilus, and cluster II formed a monophyletic group with genes from L. delbrueckii, which can grow at 45 °C [26]. The similarity between d-LDH2 and d-LDH3 was only 47%, indicating that the genes in cluster I are paralogs of the genes in cluster II. Both the phylogenetic analysis and the levels of

Conclusions

Three genes for d-LDHs were retrieved from the genome of L. jensenii and then successfully expressed in E. coli. The three enzymes, originating from the same species, were clustered into two different groups and showed different characteristics, suggesting that d-LDH genes with distinct activities can be found through genome-data mining, including the genomes of unculturable organisms or environmental metagenomes. The searching allowed the identification and subsequent characterization of a

Acknowledgments

This research work was supported by the R&D program of MKE/KEIT (10031717) and Kwangwoon University Grant for Professor Research Year (2012).

References (36)

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These authors contributed equally to this work.

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