Glycerol metabolism in the methylotrophic yeast Hansenula polymorpha: phosphorylation as the initial step

In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg²⁺ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent K _m=1.0 mM) and ATP (apparent K _m=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.Abbreviations DHA dihydroxyacetone - G3P glycerol-3-phosphate - EMS ethyl methanesulphonate - MTT 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulphate - FBP fructose-1,6-bisphosphate     

Dihydroxyacetone metabolism in <Emphasis Type="Italic">Salinibacter ruber</Emphasis> and in <Emphasis Type="Italic">Haloquadratum walsbyi</Emphasis>

The extremely halophilic bacterium Salinibacter ruber inhabits saltern crystallizer ponds worldwide, together with the square archaeon Haloquadratum walsbyi. Cultures of Salinibacter have been shown to convert up to 20% of the glycerol added to a not previously characterized overflow product. We here identify this product of incomplete glycerol oxidation by Salinibacter as dihydroxyacetone. Genomic information suggests that H. walsbyi possesses an efficient uptake system for dihydroxyacetone, and we show here that dihydroxyacetone is indeed metabolized by Haloquadratum cultures, as well as by the heterotrophic prokaryotic community of the saltern crystallizer ponds in Eilat, Israel, dominated by Haloquadratum-like cells. In the absence of glycerol, Salinibacter also takes up dihydroxyacetone. Degradation of glycerol, produced in hypersaline lakes as an osmotic solute by the green alga Dunaliella salina may thus involve dihydroxyacetone as an intermediate, which can then be taken up by different types of heterotrophs present in the environment.     

Metabolic Interactions Between Glucose, Glycerol, Alanine and Acetate in Leishmania braziliensis panamensis Promastigotes

¹³C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of ¹⁴CO₂ production from ¹⁴C-labeIed substrate. Cells incubated with [2-¹³C]glycerol released acetate, succinate and D-lactate in addition to CO₂. Cells incubated with acetate released only CO₂. More succinate C-2/C-3 than C-l/C-4 was released from both [2-¹³C]glycerol and [2-¹³C]glucose, indicating that succinate was formed predominantly by CO₂ fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of ¹⁴CO₂ production from [l,(3)-¹⁴C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased ¹⁴CO₂ production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of ¹⁴CO₂ production from [U-¹⁴C]- and [l-¹⁴C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool.     

Sugar metabolism in the extremely halophilic bacterium Salinibacter ruber

Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by a small number of sugars (glucose, maltose, starch at 1 g l(-1)). Glucose consumption starts after other substrates have been depleted. Glucose metabolism proceeds via a constitutive, salt-inhibited hexokinase and a constitutive salt-dependent nicotinamide adenine dinucleotide phosphate (NADP)-linked glucose-6-phosphate dehydrogenase. Glucose dehydrogenase and fructose-1,6-bisphosphate aldolase activity could not be detected. It is therefore suggested that Salinibacter metabolizes glucose by the classic Entner-Doudoroff pathway and not by the Embden-Meyerhof glycolytic pathway or by the modified Entner-Doudoroff pathway present in halophilic Archaea of the family Halobacteriaceae, in which the phosphorylation step is postponed. However, activity of 2-keto-3-deoxy-6-phosphogluconate aldolase could not be detected in extracts of Salinibacter cells, whether or not grown in the presence of glucose.     

Glycerol production from sugars with phosphoglycerate mutasedeficientSaccharomyces cerevisiae partially resistant to glucose repression

Summary Mutants partially resistant to the repressive effect of glucose have been isolated from aSaccharomyces cerevisiae strain totally deficient in phosphoglycerate mutase activity (EC 5.4.2.1) by a selection procedure involving the catabolite-repressive effect of 5-thio-d-glucose (5TG). These mutants are able to resist glucose concentrations up to 15 g L^–1 and exhibit several non-repressed metabolic pathways such as gluconeogenesis, glyoxylic shunt or mitochondrial respiratory chain. Moreover, when these mutants are grown in aerobiosis on ethanol and glucose as sole substrates, glucose is mainly converted into glycerol in order to maintain a normal redox balance. Optimal glucose and oxygen concentrations have been defined for resting cells in order to obtain a glycerol yield from glucose close to 100%. The physiological characteristics of one of these mutants led us to consider an application of this yeast strain in reducing the ethanol content of wines previously lowered in ethanol content by physical processes.     

Sensitivity of Haloquadratum and Salinibacter to antibiotics and other inhibitors: implications for the assessment of the contribution of Archaea and Bacteria to heterotrophic activities in hypersaline environments

Antibiotics and bile salts have been used to differentiate between heterotrophic activity of halophilic Archaea and Bacteria in saltern ponds. In NaCl-saturated brines of crystallizer ponds, most activity was attributed to Archaea. Following the recent isolation of Haloquadratum, the dominant archaeon in the salterns (reported to be sensitive to chloramphenicol and erythromycin), and the discovery of Salinibacter, a representative of the Bacteria, in the same ecosystem, reevaluation of the earlier data is required. The authors measured amino acid incorporation by Haloquadratum and Salinibacter suspended in crystallizer brine to investigate the suitability of antibiotics and bile salts to distinguish between archaeal and bacterial activities. The amino acid uptake rate per cell in Salinibacter was two orders of magnitude lower than that of Haloquadratum under the same conditions. Salinibacter was inhibited by chloramphenicol, erythromycin, and deoxycholate, but not by taurocholate. Erythromycin did not inhibit incorporation by Haloquadratum, but moderate inhibition was found by chloramphenicol at 10-50 microg mL(-1). Deoxycholate was highly inhibitory, but only partial inhibition was obtained in the presence of 25 microg mL(-1) taurocholate. Inhibition by chloramphenicol and taurocholate increased with increasing salt concentration. Erythromycin and taurocholate proved most valuable to differentiate between archaeal and bacterial activities in saltern brines.     

Kinetically controlled synthesis of monoglyceryl esters from chiral and prochiral acids methyl esters catalyzed by immobilized Rhizomucor miehei lipase

Partial acylation of only one primary hydroxyl group of glycerol generates a chiral center at position 2. Rhizomucor miehei lipase (RML) catalyzes the kinetically controlled transesterification of different aromatic carboxylic acids methyl esters with glycerol. High synthetic yields of glyceryl esters (around 70-80%) were obtained even in the presence of significant concentrations of water (from 5% to 20%). After a long incubation of the reaction mixture in the presence of the biocatalyst only pure free acid was obtained. Other lipases (from Geobacillus thermocatenulatus and from Thermomyces lanuginose) also catalyzed similar kinetically controlled transesterifications although less efficiently. RML immobilized on Sepharose-Q showed a high activity and specificity, compared to the immobilization by other techniques, only producing monoglyceryl esters with all substrates. In particular, monoglyceryl-phenylmalonate product was synthesized in 82% overall yield and >99% diastereomeric excess at pH 7.0 and 37 °C and 90% glycerol.     

The role of glycerol in the nutrition of halophilic archaeal communities: a study of respiratory electron transport

Abstract Respiratory electron transport activity in the Dead Sea and saltern crystallizer ponds, hypersaline environments inhabited by dense communities of halophilic archaea and unicellular green algae of the genus Dunaliella , was assayed by measuring reduction of 2-( p -iodophenyl)-3( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Typical rates obtained were in the order of 5.5–17.7 nmol INT reduced h ⁻¹ per 10⁶ cells at 35 ° C. In Dead Sea water samples, respiratory activity was stimulated more than two-fold by addition of glycerol, but not by any of the other carbon compounds tested, including sugars, organic acids, and amino acids, or by addition of inorganic nutrients. Stimulation by glycerol had a half-saturation constant of 0.75 μM. A similar respiratory activity was also found when Dead Sea water samples were diluted with distilled water and incubated in the light. As Dunaliella cells did not reduce INT, it is suggested that photosynthetically produced glycerol leaking from the algae is the preferred carbon and energy source for the development of halophilic archaea in hypersaline environments. In samples from saltern crystallizer pond stimulation of INT reduction by glycerol was much less pronounced, probably because the community was less severely carbon-limited.     

Dihydroxyacetone kinase from a methylotrophic yeast,Hansenula polymorpha CBS 4732

Dihydroxyacetone (DHA) kinase was purified to electrophoretic homogeneity from methanol-grown Hansenula polymorpha CBS 4732. The enzyme was a dimer with a molecular weight of 150,000, and had an isoelectric point of 4.9. The enzyme was active toward DHA, and D- and L-glyceraldehydes as phosphorylation acceptors, and only ATP served as a donor. ADP inhibited the enzyme at a physiological concentration. Magnesium ion was essential for the activity and stability. Some other divalent cations can substitute in part the magnesium ion. The DHA kinases found in cells grown on methanol and glycerol were immunologically identical, but were different from those of other methylotrophic yeasts as shown by immunotitration. A mutant (204D) derived from the yeast, which could not grow on methanol or DHA but could so on glycerol, was deficient in DHA kinase. Glycerol kinase activity was found in glycerol-grown 204D cells as well as the parent strain.Abbreviation DHA dihydroxyacetone     

Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes

Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines. It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures. Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation. This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization. The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed. Inactivation of the protein by autolysis did not occur. This paper reports the use of a series of polyol osmolytes to stabilize PA. Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent. Addition of NaCl to glycerol solution further enhanced the thermal stability of PA. An increase in the T(1/2) value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20 degrees C was observed. The half-life (t(1/2)) of PA thermal inactivation at 40 degrees C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control. This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax.     

Heterologous expression of Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) and glycerol dehydrogenase gene (ZrGCY1) in Saccharomyces cerevisiae

We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Deltagpd2Delta). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.     

Acetylation of glycerol to biofuel additives over sulfated activated carbon catalyst

Oxygenated fuel additives can be produced by acetylation of glycerol. A 91% glycerol conversion with a selectivity of 38%, 28% and 34% for mono-, di- and triacetyl glyceride, respectively, was achieved at 120 °C and 3 h of reaction time in the presence of a catalyst derived from activated carbon (AC) treated with sulfuric acid at 85 °C for 4h to introduce acidic functionalities to its surface. The unique catalytic activity of the catalyst, AC-SA5, was attributed to the presence of sulfur containing functional groups on the AC surface, which enhanced the surface interaction between the glycerol molecule and acyl group of the acetic acid. The catalyst was reused in up to four consecutive batch runs and no significant decline of its initial activity was observed. The conversion and selectivity variation during the acetylation is attributed to the reaction time, reaction temperature, catalyst loading and glycerol to acetic acid molar ratio.     

Regulation of glycerol metabolism in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> NBRC12010 under electrochemical conditions

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD⁺/NADH. At a NAD⁺/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD⁺/NADH. Exogenous NADH was oxidized to NAD⁺ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.     

Dual pathways of glycerol assimilation in Klebsiella aerogenes NCIB 418

Klebsiella aerogenes NCIB 418 assimilates glycerol via alternative pathways: one involves a glycerol kinase with a high affinity for glycerol (apparent K _m=1–2×10^–6 M), and the second a glycerol dehydrogenase with a much lower affinity for its substrate (apparent K _m=2–4×10^–2 M).In variously-limited chemostat cultures, one or the other pathway predominated. Thus, aerobic carbonlimited organisms contained only the glycerol kinase pathway whereas aerobic sulphate-limited or ammonia-limited organisms (grown on glycerol) used only the glycerol dehydrogenase pathway. Anaerobic cultures invariably contained glycerol dehydrogenase, and glycerol kinase was absent.Washed suspensions of aerobically-grown organisms oxidized glycerol with kinetics similar to that of the particular enzyme (the primary enzyme of the assimilatory pathway) which they possessed, thus indicating a close association between these two enzymes and the uptake process. But a supply of exogenous glycerol was not a prerequisite for the synthesis of either glycerol kinase or glycerol dehydrogenase, and nor was molecular oxygen the key factor in effecting modulation between the alternative pathways of glycerol metabolism, as had been previously suggested.The physiological significance of dual pathways of glycerol assimilation is discussed.     

Purification and characterization of glycerol-3-phosphate dehydrogenase (NAD+) in the salt-tolerant yeast Debaryomyces hansenii

The NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of the salt-tolerant yeast Debaryomyces hansenii was purified by poly(ethylene glycol) precipitation and a combination of chromatographic procedures. The enzyme existed in two forms with different ionic characters and specific activity. On SDS-polyacrylamide gel electrophoresis, both forms yielded one predominant band with an apparent molecular weight of 42,000. The specific activity of the enzyme was dependent on the concentration of the enzyme and on the ionic strength of the dissolving medium. All ions tested stimulated the enzyme activity in the ionic strength range 0-100 mM, with glutamate yielding the highest activity. Above these concentrations, the dehydrogenase showed high tolerance for glutamate in concentrations up to 0.9 M, whereas malate, sulfate and chloride were inhibitory. Enzyme activity showed little sensitivity to the type of cation present and was only slightly affected by 5 M glycerol. The true Km values for the substrates were 6.6 microM for NADH, 130 microM for dihydroxyacetone phosphate, 0.3 mM for NAD and 1.2 mM for glycerol-3-phosphate, and the enzyme showed specificity for these four substrates only. It is proposed that the enzyme functions in cellular osmoregulation by providing glycerol 3-phosphate for the biosynthesis of glycerol, the main compatible solute in D. hansenii, and that the enzyme is well adapted to function in yeast cells exposed to osmotic stress.     

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA dihydroxyacetone - DHAP dihydroxyacetone phosphate - G3P glycerol 3-phosphate - GAP glyceraldehyde 3-phosphate - 3-PGA 3-phosphoglycerate - 2-PGA 2-phosphoglycerate - 2,3-DPGA 2,3-diphosphoglycerate - PEP phosphoenolpyruvate - DH dehydrogenase - GK glycerol kinase - DHAK dihydroxyacetone kinase - TIM triosephosphate isomerase - PGK 3-phosphoglycerate kinase - PK pyruvate kinase - LDH lactate dehydrogenase - DTT dithiotreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - BV²⁺/BV⁺ oxidized/reduced benzylviologen - PMS phenazine methosulfate - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide     

Effect of equilibration period on the viability of frozen-thawed mouse morulae after rapid freezing

Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.     

Distribution of glycerol dehydrogenase and glycerol kinase activity in halophilic archaea

Abstract A constitutive NAD⁺-dependent glycerol dehydrogenase activity was detected in Halobacterium salinarium and Halobacterium cutirubrum . Optimal activity was found at 3 M KCl and pH 8–10. No glycerol dehydrogenase activity could be demonstrated in representatives of the genera Haloferax and Haloarcula , even when grown in the presence of glycerol, or in Halobacterium saccharovorum and Halobacterium sodomense . Glycerol kinase activity was shown to be present constitutively in all halophilic archaea examined. The finding that glycerol dehydrogenase is found only in part of the halophilic arachaea makes dihydroxyacetone an improbable candidate as the precursor for the glycerol moiety of halobacterial lipids.     

产甘油假丝酵母产甘油关键酶基因在酿酒酵母中的表达

甘油是一种极其理想的耐高渗透压介质。利用PCR方法,从产甘油假丝酵母WL2002-5中扩增出了2个产甘油的关键酶基因GPD和GPP,分别编码3-磷酸甘油脱氢酶（glycerol 3-phosphate dehydrogenase, GPD）和3-磷酸甘油磷酸酶（glycerol 3-phosphate phosphatase, GPP）。利用T-Vector在Escherichia coli JM109中克隆得到大量的GPD和GPP基因,并成功构建了重组质粒pYX212-GPD和pYX212-GPP;通过LiAc转化法将重组质粒导入酿酒酵母Saccharomyces cerevisiae W303-1A。初步实验结果表明：发酵过程中pYX212-GPD/S. cerevisiae W303-1A的生物量高于pYX212-GPP/S. cerevisiae W303-1A和野生型S. cerevisiae W303-1A;发酵72h后,pYX212 GPD/S. cerevisiae W303-1A发酵液中甘油含量大约为12mmol/L,明显高于野生型S. cerevisiae W303-1A的甘油含量,而pYX212-GPP/S. cerevisiae W303-1A与野生型S. cerevisiae W303-1A在甘油含量上相差不大,均只有4mmol/L 左右。