Progress,in,Enzymatic,Production,of,Trehalose
Longxiang SONG Shaojie YANG Hongling LIU Tengfei WANG
Abstract Trehalose is a non-reducing disaccharide， and widespread in nature. It is a typical stress metabolite that can protect macromolecules such as proteins in organisms under extreme conditions. Therefore， trehalose has been widely used in food， medicine and cosmetics. Trehalose is extracted from yeast or synthesized by chemical method. Due to the high cost of traditional methods， trehalose is mainly produced by enzymatic methods. There are mainly three pathways：
TPS/TPP， TreY/TreZ and TreS. These enzymatic systems are expressed mainly through heterologous expression.
Key words Trehalose; Application; Enzymatic production
Physicochemical Properties of Trehalose
In 1832， Wiffers H. A. L.， a German chemist， discovered trehalose in the ergot fungus of rye[1]. In 1859， Berthelot M.， a French chemist， first isolated trehalose from trehala Manna in weevil and named it[2]. Trehalose is a nonreductive disaccharide， which widely exists in organisms such as algae， yeast， mildew fungi， edible fungi， shrimp， insects， higher plants and so on. It usually exists in the form of dihydrate trehalose in nature and is not easy to be hydrolyzed by acid. Trehalose has three optical isomers. α，α is the most common trehaolse found in nature， while α，β and β，β are rarely found in nature and are mostly synthesized artificially.
Trehalose is a white crystal with two crystal water. Its molecular formula is C12H22O11·2H2O and the molecular weight is 378.33. It is composed of two glucose residues through the half acetal hydroxyl phase junction stage. It has weak sweetness and can dissolve in water and hot alcohol， insoluble in ethyl ether. It is poisonless and harmless and has stable chemical properties （Table 1）. It can also be hydrolyzed into glucose by enzymes and be used when trehalose is ingested.
Protective Mechanism and Application of Trehalose
As early as 1998， researchers showed that trehalose protects proteins in living cells at high temperatures， keeping them in their natural state[6]. As more research was carried out， it was found that trehalose has excellent antiseptic property and high resistance to adversity. It has the function of protecting biological cells and bioactive substances from being destroyed under adverse environmental conditions such as dehydration， drought， high temperature， freezing， high osmotic pressure and toxic reagents. Many trehalose-containing plants and animals remain their active after complete drying. As soon as they encounter water， they come back to life.
The biological protection mechanism of trehalose is generally considered to be that the biological protection mechanism of trehalose can strongly soothe the water molecules， and it can share the binding water with the ester membrane or trehalose itself can replace the membrane binding water to prevent the degeneration of the protein membrane. Zhang[7]conducted infrared spectroscopy analysis on the mixture of trehalose and lecithin， and the results showed that trehalose could form hydrogen bonds with cell membrane components to replace water， thus protecting the stability of cell membrane.
As a stress metabolite， the content of trehalose varies with the environmental conditions[8]. Microorganisms accumulate trehalose in high saltnity environmental to make themselves have high osmotic pressure tolerance. Escherichia coli acquires osmotic pressure resistance by accumulating trehalose in high-salt environments[9]. Saccharomyces cerevisiae did not show significant content of trehalose in cells at 28 ℃. When the temperature rose from 28 to 40 ℃， content of trehalose increased 5-9 times， and when temperature dropped to 28 ℃， trehalose content also decreased[10].
At present， trehalose has been widely used in food， cosmetics and medicine because of its protective effect. Because trehalose has many advantages such as low sweetness， high solubility， low cariogenicity and high stability， it is usually used as a food additive. In cosmetic， the main effects of trehalose are to protect wet， resist radiation， resist skin consenescence and so on. Trehalose has also been found to alleviate Huntington disease in a mouse model， which is mediated by polyglutamine[11]. Trehalose also protects vaccines， enzymes and animal cells from freeze-drying at room temperature[12].
Trehalose is the anti-metabolite of many organisms and plays an important role in bioresistance. As trehalose can protect protein lipid membrane and cells in severe environments， protect the integrity of biofilm and maintain biological activity[13-14]， it plays an important role in pharmaceutical food and cosmetics.
Traditional Production of Trehalose
Although trehalose is widely distributed， its content is low in natural organisms. The traditional production method is mainly extracted from yeast， because the content of trehalose is large in yeast that the content is up to 20% of the dry weight of yeast. But it is expensive which limits the large-scale production. In addition， there is another method of production-chemical method. Chemical method was developed as early as 1954：
α，α-trehalose was synthesized using the ethylene oxide addition reaction between 2，3，4，5-tetra-O-acetyl-D-glucose and 3，4，6-tri-O-acetyl-1，2-anhydro-D-glucose[15]. Many synthetic methods of trehalose and its analogues and derivatives have been reported （Fig. 1）， but it is also difficult to apply to industrial production. Because it has great disadvantages， such as low productivity， many by-products and difficult to purify[15-16].
Because traditional methods were not suitable for mass production， scholars began to look for other more efficient ways to produce trehalose.
Enzymatical Production of Trehalose
With the further research of trehalose， some methods of biosynthesis of trehalose have been reported， which mainly include three approaches， TPS/TPP （or OtsA/OtsB） pathway， TreY/TreZ pathway and TreS pathway[19-21].
First， the most extensive approach in nature is the TPS/TPP pathway. This pathway was first discovered in yeast in 1958， and has been found in bacteria， fungi， plants， insects and many other organisms[22]. Trehalose was previously thought to be found only in some drought-tolerant plants. Later， some researchers found that the TPP/TPS homologous genes isolated from plants could well repair the trehalose synthesis defect strains of yeast， indicating that plants could synthesize trehalose through TPP/TPS pathway[23]. This pathway involves two enzymes， trehalose 6-phosphate synthase （TPS or OtsA） and trehalose 6-phosphate phosphorylase （TPP or OtsB）. TPS first converts the glucose unit from UDP or GDP-glucose to glucose-6-phosphate to obtain the intermediate trehalose-6-phosphate， which is then hydrolyzed in trehalose by TPP [24-26]（Table 2）. Leandro Padilla heterogeneously expressed OtsA and OtsB from E. coli in Corynebacterium glutamicum and the activity of OtsAB pathway and trehalose production in recombinant strain were successfully increased[24].
The second method contains another two enzymes， maltooligosyltrehalose synthase （TreY/MTSase） and maltooligosyltrehalose trehalohydrolase （TreZ/MTHase）. This pathway exists mainly in archaea. TreY acts on maltodextrin or starch， reducing the α-1，4-bond at the disaccharide end to α-1，1-bond， and then TreZ releases trehalose from the low molecular weight maltodextrin[28-29]. TreY is a key enzyme in this pathway. Chen increased the yield of trehalose by directed evolution and site directed mutation on TreY from Arthrobacter ramosus[30]. In fact， a protein， TreX， is involved in this pathway. It codes for a glycogen debranching activity[22].
The last is the direct method， which uses only trehalose synthase （TreS）. TreS takes maltose as the substrate directly and isomerizes it into trehalose with no by-product or by-product except glucose[31-34]. This method is simple， practical and low cost which is suitable for industrial production of trehalose （Fig. 2）. This pathway mainly exists in bacteria. TreS was first discovered in Pimelobacter sp. R48. So far， TreS has been found in a variety of bacteria， including Thermomonospora curvata， Thermus aquaticus， Picrophilus torridus， Pseudomonas stutzeri， Pseudomonas putida， Arthrobacter auresce， Enterobacter hormaechei， Meiothermus ruber[38].
The above pathways can also coexist in a single strain， for example， Corynebacterium glutamicum[24]. With the development of DNA recombination technology， more and more researchers have shifted their research goat to improve trehalose production by using heterologous expression. Currently， the most commonly used engineered strain is E. coli. Wang fused the β-amylase gene of Clostridium thermosulfuro with trehalose synthase gene of Thermus thermophilus and successfully expressed in E. coli [35]. Li combined trehalose synthase genes from 5 different sources with 4 different vectors and expressed them in E. coli DE3. It was found that trehalose synthase gene from T. thermophilus HB27 and pET-22b could be highly expressed in E. coli[36]. Zheng[37]expressed three treS genes from different sources in E. coli BL21 respectively， and found that the treS from P. tutzeri A1501， gene ID 5095109， had the best protein transformation ability， and the purified enzyme activity was 32.8 U/mg. In addition， in order to avoid the complicated operation caused by ultrasonic crushing， they used chloroform to permeate the recombinant E. coli and carried out whole-cell catalysis. After the optimized condition， the conversion reached a balance. The concentration of trehalose was 96.0 g/L and conversion rate was 16.0 g/h.
Because trehalose is mostly used in food， medicine and cosmetics， a safer way to produce it is needed. To this end， researchers have expressed trehalose synthase genes in Bacillus subtilis， a food safety strain. Liu[38]selected TreS from Pseudomonas malodorius， expressed it in B. subtilis， and modified it through saturation mutation （V407M and K490L）， and found that the conversion rate and enzyme activity of the modified TreS were improved. Meanwhile， several strong promoters of B. subtilis were selected and tandem to control the expression of the treSV407M-K490L gene， and it was found that when the Psrf-cry3a promoter was used， the enzyme activity reached 5 800 U/g dry weight. Wang [40]fused the signal peptide with TreS and expressed them in B. subtilis. Guided by the signal peptide， TreS was secreted to the extracellular.
Conclusion
In the early 1990s the cost of 1 kg of commercialized trehalose could reach $700. With the in-depth study of trehalose， its production cost has been efficiently reduced. For now， high added value applications like pharmaceuticals can afford trehalose， but it"s not enough for industries like food [12]. At present， the production method of trehalose is mainly enzymatic method. And most of the researches aim to improve the enzyme activity of trehalose synthase system， so as to increase the production of trehalose and reduce the cost. This is of great significance to the industrial production of trehalose. With the in-depth study of trehalose， trehalose will have a more extensive application prospects， but also bring greater convenience and benefits to people.
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