One-step purication and characterization of two novel Thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278

A newly screened cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) from fermentation solution were recovered by modied non-denaturing active gel electrophoresis and gel ltration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃ , and the enzymes were stable under 70 ℃ . The optimal pH of the puried enzymes was 6.0, and the enzymes were stable between pH 4–10. Km and Vmax values of 2.76 mg/mL, 20.6 U/mg for pNPG. Thin-layer chromatography and high-performance liquid chromatography analysis showed that cellobiose BG FH1and BG FH2 had hydrolysis activity and can hydrolyze cellobiose into glucose. In addition, both enzymes also exhibited transglycoside activity, which can use low molecular weight monosaccharides to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 can produce heat-resistant β-glucosidase with both hydrolytic activity and transglycosidic activity, and has potential application value in bioethanol and papermaking industries.


Introduction
Lignocellulose is a linear polysaccharide linked by D-glucose via β-(1,4)-glycosidic bonds, and is the most abundant renewable resource on earth (Zhang et al., 2010;Kovacs et al., 2009;Sánchez and Cardona, 2008). Lignocellulose can eventually be degraded into glucose under the synergy of the cellulase system: endoglucanase (EC 3.2.1.4) randomly acts on the non-crystalline region inside the cellulose molecule to produce glucose and short ber oligosaccharides sugar; Exoglucanase (EC 3.2.1.91) hydrolyzes β-1,4-glycosidic bonds from outside to inside along the non-reducing end of cellulose to release cellooligosaccharides, cellobiose or glucose; β-glucosidase (EC 3.2 .1.21) hydrolyzes cellobiose or other soluble cellobiose and cellooligosaccharides into glucose (Gomes et al., 2018;Fan et al., 2016;Arantes and Saddler, 2010 ). β-glucosidase plays an important role in the hydrolysis of cellulose. The intermediate products of cellulose hydrolysis, such as cellobiose, cellooligosaccharides, have a strong inhibitory activity on activity of exoglucanase and endoglucanase. β-glucosidase hydrolyzes cellobiose and cellooligosaccharides to produce glucose, reducing the inhibition of these intermediate products on exoglucanase and endoglucanase and improving the sacchari cation rate of cellulose enzymes, therby playing a key role in the degradation of cellulose (Gomes et al., 2018;Kamila et al., 2016;Chauve et al., 2010;Ikeda et al., 2006;Tanaka et al., 2006;Wen et al., 2005).
β-glucosidase is an important industrial enzyme that plays an important role in a variety of biotransformations. It has been used in many bioprocesses, including the processing of biofuels, paper industry, textile industry, waste, and food (Pei et al., 2012;Tian et al., 2010;Bayer et al., 2008;Han and Chen, 2008;Rubin 2008;Villena et al., 2007;Zaldivar et al., 2001).
Thermophilic fungi can produce a large amount of hydrolytic enzymes that hydrolyze cellulosic substances. The produced hydrolytic enzymes have good catalytic performance, high yield, and good stability, and are a promising industrial enzyme. (Zhang et al., 2014;Haven and Jørgensen, 2013;Prawitwong et al., 2013;Pei et al., 2012).
Screening of strains producing high β-glucosidase activity is very important for the comprehensive utilization of cellulose resources and their application in other elds (Miettinen-Oinonen et al., 2004;Saloheimo et al., 1997). Xylanase, endoglucanase, and other cellulose hydrolyzing enzymes from Fusarium sp. have strong cellulose degradation ability, and the synergistic effect of these enzymes can convert cellulose into ethanol Gómez-Gómez et al., 2001;Kumar et al., 1991).
In our previous study, a heat-resistant cellulase-producing F. chlamydosporum strain HML278 was screened from the virgin forest samples in Guangxi, China. We for the rst time identi ed that this strain can produce and secrete three major enzyme components of cellulase system, endoglucanase, microcrystalline cellulose, and β-glucosidase and xylanase (Qin et al., 2010). In this study, two new β-glucosidases with hydrolytic activity and transglycosidic activity from F. chlamydosporum HML278 were rapidly isolated and puri ed by using improved non-denaturing active gel electrophoresis combined with gel ltration chromatography. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable below 70 ℃. The new identi ed enzymes may have great potential in industrial applications of bioethanol, papermaking, etc.

Materials And Methods
Production of cellullase by solid-state fermentation and enzymatic activity test Strain: F. chlamydosporum HML278 used in this study was maintained on PDA medium at 4℃ in Guangxi Colleges Universities Key Laboratary of Exploitation and Utilization of Microbial and Botanical Resources.
Production of cellullase by solid-state fermentation: The screened cellulase-producing strain grown on PDA slant was washed off with 10 mL of physiological saline to make a spore liquid, and 10 7 spores were transferred to the solid medium for second screening of cellulase. To make the solid medium for cellulase screen, 6 g bagasse, 4 g bran, and 30 mL Mandels nutrient solution (Kwon et al., 1994) were mixed in 500 ml erlenmeyer ask. The ask was ipped twice a day, and the strain was grown for 4 days at 30 °C. 200 mL of sterile ddH 2 O was added to the culture, and was further leached at 40 °C in a constant temperature water bath for 1 hour. The culture was ltered with four layers of gauze, and centrifuged at 6000 r/min for 10 min. The supernatant containing crude enzymes was collected and stored at 4 °C until use (Qin et al., 2010).
Detection of β-Glucosidase enzyme activity: 0.02 M citric acid-sodium citrate buffer solution (pH 4.8) was used to prepare 1% salicin (Fluka Chemical Corp, USA) solution substrate. 0.05 mL of enzyme solution with appropriate concentration was mixed with 1mL of 1% salicin solution, and the reaction was carried out at 60 °C for 30 min. 3 mL of DNS reagent was added to stop the reaction. The reaction solution was boiled for 6 min, followed by cold water bath. The absorbance was measured at 540 nm. The amount of enzyme that produces 1 μmol of glucose per minute was de ned as 1 unit of enzyme activity (U) (Shoemaker and Brown, 1978).

Detection of soluble total protein
The protein concentration was measured at 595 nm according to the Bradford method (Bradford, 1976) by using a Bradford Protein Assay Kit (Beyotime Institute of Biotechnology, China) following the manufacture's instruction.
Puri cation of β-glucosidase All of puri cation steps were performed at 4 °C.
Active recovery of non-denaturing gel electrophoresis: The non-denaturing gel consisting of 8% separation gel and 4% stacking gel was run at 50 V constant voltage at 4 °C. After electrophoresis, the activity of β-glucosidase in the gel was detected by staining of gel with speci c substrate (Kwon et al., 1994) containing 0.03% FeCl 3 and 0.1% aescin.
After staining for 1 min at 30 °C, the gel was immediately rinsed with distilled water to stop the reaction. The active protein band with black precipitation was cut off, and grinded in a pre-cooled mortar. The sample was leached with citric acid-citrate buffer (20 mM, pH 4.8) at 4 °C for 12 h, and centrifuged at 4000 r/min for 20 min in a 5000 Da ultra ltration tube for concentration and desalting.
The enzyme solution was further puri ed by HiPrep 16/60 Sephacryl S-200 h\High Resolution gel ltration chromatography column, using a BioLogic DuoFlow Path nder 80 puri er system (pressure 73 psi). The enzyme was eluted by using elution buffer containing 0.05 mol/L PBS and 0.15 mol/L NaCl (pH 7.2) at the ow rate of 1 mL/min. The enzyme activity of the puri ed protein was detected referring to the enzyme activity rapid detection plate of βglucosidase, and the protein purity was detected by using SDS-PAGE.

SDS-polyacrylamide gel electrophoresis SDS-PAGE
12% resolving gel was used in SDS-polyacrylamide gel electrophoresis, and the gel was stained with Coomassie Brilliant Blue R250. The molecular weight of puri ed proteins was assessed by comparing the relative mobility of puri ed protein with low molecular weight standard protein (Laemmli, 1970).

Zymogram analysis of puri ed enzyme
The collected extracellular enzyme solution of HML278 was subjected to non-denaturing protein gel electrophoresis with pH 8.3 electrophoresis buffer at 4 ℃ by using 50 V constant voltage. The separation gel and stacking gel was made by 8% acrylamide and 4% acrylamide, respectively. After the electrophoresis, the acrylamide separation gel was cut and partly stained with silver, and the other part was stained with speci c substrates of different cellulases.
To analyze the activity of β-glucosidase from cut gel, the gel was active stained with staining solution containing 0.1% escin (Sigma) and 0.03% ferric chloride (Sigma) for 5 minutes at 30 ℃. The protein with β-glucosidase activity will catalyze the substrate to produce a yellow-black product (Kwon et al., 1994).
Analysis on the hydrolysis activity of puri ed β-glucosidase Experiments for HML0366 β-glucosidase enzyme hydrolysis activity and transglycoside-mediated synthesis of gentiobiose.
β-glucosidase hydrolysis assay: 10 mL of 1 (m/v) cellobiose dissolved in citrate buffer (50 mM, pH 4.8) was used as a substrate, and 2 mL of enzyme solution was added to react at 30 °C for 30 min.
TLC method for detecting sugar components (Jo et al., 2003): Silica thin-layer chromatography detection was utilized. Expanding agent: n-butanol: ethyl acetate: ammonia: water = 6: 3: 3: 1 (v/v). Developer: A: 1g aniline + 25 mL acetone, B: 1 mL dianiline + 25 mL acetone. After mixing A and B, 5 mL 85% phosphoric acid was added and mixed well. After the chromatography, the plate was blown dry and color developer was sprayed, and dried at 120 °C for 10 minutes to develop color.
Cellobiose was dissolved in 20 mM citrate buffer (pH 4.8), and enzyme solution was added at 100: 1 (v/v), and reacted at 30 °C for 3 h. The product was detected by thin-layer chromatography (Jo et al., 2003;Qin et al., 2011).
Detection and identi cation of proteins by tandem time-of-ight mass spectrometry Identi cation of puri ed enzymes by tandem time-of-ight mass spectrometry: After SDS-PAGE, the β-glucosidase band was cut out, and the ngerprints of peptide fragments were obtained after scanning analysis by time-of-ight mass spectrometry (4800 Proteomics Analyzer, Applied Biosystems, USA), and the data was analyzed by using the Mascot software to query and identify puri ed enzymes on the SWISS-PROT database (Scheibner et al., 2008;Lee et al., 2007).

Enzymatic properties of puri ed enzyme
The effect of temperature on the enzyme activity and stability of β-glucosidase The de nition of relative enzyme activity: the highest enzyme activity under a certain condition of the experimental project was set to 100 , and the ratio of enzyme activity under other conditions to the highest enzyme activity was de ned as relative enzyme activity.
The enzyme activity was measured under the conditions of 30℃-90℃ in 50 mM acetate buffer to determine the optimal temperature of endoglucanase and β-glucosidase.
To determine the effect of temperature on the stability of β-glucosidase, the enzyme was further incubated in a water bath at temperatures between 40 ℃ and 90 ℃ with a gradient of 5 ℃. The enzymes were incubated at each temperature for 60 min, and the residual enzyme activity was then measured at 60 ℃.
The impact of pH for enzyme activity and stability of β-glucosidase To determine the effect of pH on enzyme activity of β-glucosidase, the following four solutions with a concentration of 50 mM were used: disodium hydrogen phosphate-citric acid buffer, pH 2.6-7.5; Tris-HCl buffer, pH7.5-pH 9.0; glycine-NaOH buffer, pH 9.0-11.0.
Under the temperature condition where the enzyme is stable, the enzyme was mixed with the buffer with a pH value ranging between 3.0 to 9.0, and the relative enzyme activities and the optimal pH value of endoglucanase and βglucosidase were determined.
The enzyme was further stored in a solution with a pH value between 3.0-11.0. After being left at 4 °C for 24 hours, it was kept at 30 °C for 3 hours. The relative enzyme activities of endoglucanase and β-glucosidase were determined at the optimum pH and temperature.
Effect of metal ions on β-glucosidase enzyme activity Different metal ions were added to the puri ed enzyme solution with a nal concentration of 2 mM, and the enzyme activity was tested. The enzyme activity was calculated according to the average value of data from three parallel experiments.

Kinetics analysis of the puri ed β-glucosidase
To determine the kinetic parameters of the enzymatic reaction of β-glucosidase, pNPG was used as the substrate and the reaction was performed under pH 4.8 at 30 ℃. The initial reaction rate was calculated, and the Km value and Vmax of the puri ed β-glucosidase was calculated by using double reciprocal plotting method (Lineweaver-Burk plot (Lineweaver and Burk, 1934).

Results
Puri cation and characterization of β-glucosidase from F. chlamydosporum HML278 fermented solution It was shown that the enzyme activity of β-glucosidase reached a maximum of 115.2 U/g after 4 days of solid bagasse culture of F. chlamydosporium HML278 enzyme activity. To purify the enzymes from fermented solution of F. chlamydosporium HML278, an anion exchange column was initially utilized, but the separation effect was not good, and there was no obvious protein peak, it was speculated that the isoelectric point may be too high. A total of 115.2 U (20mL) of crude enzyme solution was further subjected to non-denaturing gel electrophoresis without adding a comb in order to increase the sample load (Figure 1). The active gel was recovered, and subjected to gel ltration chromatography. The enzyme BG FH1was obtained after about 48 min, and BG FH2 was obtained about 64 min after running (Figure 2). SDS-PAGE analysis showed that the molecular weights of BG FH1and BG FH2 were 93 kDa and 52 kDa, respectively (Figure 3), and the recovery rates of puri cation were 4.0% and 20.0%, respectively. The foldpuri cation of BG FH1and BG FH2 was 14.0 and 28.8 (table 1), respectively and the enzyme activity for each enzy was 5.6 U/mg and 11.5 U/mg, respectively. The zymography analysis of non-denaturing electrophoresis showed that the strain produced two different β-glucosidases, and these two β-glucosidases are a single subunit enzyme ( Figure  4).
In addition, some other peptide sequences were detected by tandem time-of-ight mass spectrometry, but there was no any enzyme information in the protein database, thus these protein sequences were not identi ed.

The hydrolysis activity of BG FH1 and BG FH2
The thin-layer chromatography experiment showed that both BG HG1 and BG HG2 have hydrolytic activity, and can hydrolyze cellobiose to generate glucose. In addition, the enzymes also showed transglycoside activity and can synthesize cellotriose and cellotetraose using low molecular weight monosaccharides ( Figure 5).
High performance liquid chromatography analysis showed that BG FH1 hydrolyzed cellobiose (retention time, 10.72 min) to obtain glucose (retention time 7.36 min), and glucose could be further used as a substrate to synthesize cellotriose (retention time 12.46 min) ( Figure 6). BG FH1 transglycoside activity is stronger than other strains of the same genus (Kaya et al., 2008;Seidle et al., 2006;Seidle and Huber, 2005).
In addition, high-performance liquid chromatography analysis also showed that BG FH1can hydrolyze cellobiose into glucose and also synthesize cellotriose (Figure 6), indicating that BG FH1has transglycoside activity.
The properities of β-glucosidase isolated from F. chlamydosporum HML278 Optimum temperature and thermal stability of β-glucosidase The experimental results showed that the optimum temperature of β-glucosidases BG FH1 and BG FH2 from F. chlamydosporium HML278 was 60 ℃ at pH 5.0. The β-glucosidase exhibited good stability at temperature below 70 ℃, and the the enzyme can maintain 75% of the enzyme activity when incubated at 70 ℃ for 1 hour (Figure 7).
Enzymes that are stable at temperatures above 60 °C are de ned as heat-resistant enzymes, and these enzymes play an important role in the production of alcohol by enzymatic sacchari cation and fermentation of biomass materials (Alani et al., 2008). It has been shown that Fusarium species can produce heat-resistant cellulase (Quarantin et al., 2019;Christakopoulos et al., 1995;Kumar et al., 1991;Christakopoulos et al., 1989;Matsumoto et al., 1974;Wood, 1969;). Our study for the rst time reported that F. chlamydosporium can produce heat-resistant cellulase (Qin et al., 2010).
Chan et al. puri ed β-glucosidase DT-Bgl and showed that the enzyme exhibited the maxium activity at 70 °C. After hydrolysis of substrate into glucose, it can be further fermented to produce ethanol (Chan et al., 2016). Liew et al. puri ed a new β-glucosidase BglD5 (GH1) from Jeotgalibacillus malaysiensis, and BglD5 was stable at temperature below 65℃, which promoted the cellulase hydrolysis (Liew et al., 2018 ). Kumar found that endoglucanase and βglucosidase did not lose their enzymatic activity within 60 minutes at 80 °C. These high-temperature enzymes are suitable for application in cellulosic ethanol production . Tiwari et al. found that β-glucosidase RA10 from Bacillus subtilis was stable at a temperature of 80 ℃. The heat-stable β-glucosidase enhanced sacchari cation e ciency and thus released much higher level of glucose than previous reports. This enzyme can enhance the e ciency of hydrolysis and hydrolyze the substance of cellulose into fermentable sugar (Tiwari et al., 2017). Xia et al. found that the cellulase with good thermal stability (stable at 60 ℃) can signi cantly improve the sacchari cation e ciency of cellulose hydrolysis (Xia et al., 2016).
Thermophilic fungi can produce heat-stable enzymes It is of note that cellulose swells at high temperature, which makes it easier to break down. Thus, high temperature can promote the penetration of enzymes into materials and result in a better degradation. The screening of thermophilic fungi and the application of heat-resistant enzymes are important research directions for comprehensive applications of cellulose (Moretti et al. 2012;de Cassia Pereira et al. 2015).
The optium pH and stability of puri ed β-glucosidase at different pH conditions The β-glucosidases produced by F. chlamydosporum HML278 were relatively stable in the pH ranging from 4.0 to 10.0, and showed maximum activity pH 6.0 (Figure 8). Christakopoulos et al. screened a strain of Fusarium oxysporum and which had a optimum pH of 4.5 (Christakopoulos et al. 1995). Matsumoto et al. screened a Fusarium moniliforme strain and the β-glucosidase produced by this strain was stable at pH between 4.0 to 11.0, whereas the enzyme with a wide pH tolerance range usually has broader applications (Matsumoto et al. 1974). Since F. chlamydosporum has a wode pH tolerance range, it may have greater application potential (Christakopoulos et al. 1995;Christakopoulos et al., 1989;Matsumoto et al. 1974;Wood 1969).
Effect of metal ions on the β-glucosidase puri ed from F. chlamydosporum HML278 Metal ions are often used as activators or inhibitors in the catalytic reaction of enzymes (Grasso et al., 2012). Therefore, adding appropriate metal ions to the enzyme reaction system can improve the catalytic e ciency of the enzyme.
It was observed that all divalent metal ions had different effect on the enzyme activity. The bivalent ions Hg 2+ and Co 2+ completely inhibited enzyme activity. Hg 2+ can interact with cysteine residues in sulfhydryl bonds (Stricks and Kolthoff, 1953). It reacts with cysteine residues, especially in -SH group, and can change the tertiary structure of the protein (Lee et al., 2018). It was speculated that the active site may contain sulfhydryl groups, and these sulfhydryl groups participate in the catalysis and are essential for maintaining the structure of the enzyme (Joo et al., 2009).
The divalent cobalt ion forms a complex with various amino acids, and binding of the cobalt ion to active site of the enzyme is irreversible, completely inhibiting the activity of the enzyme. Other ions, such as Mg 2+ , Mn 2+ , Ca 2+ , Na + , Cu 2+ , and Fe 3+ also tend to form metal complexes with proteins, which ultimately affect enzyme activity by changing protein structure (Shrivastava et al., 2017).
Feng reported that Ca 2+ increased β-glucosidase Bgl3A activity by 20% (Feng et al., 2015). Xie reported that Ca 2+ at a concentration of 5 mM increased β-glucosidase activity by 58% (Xie et al., 2015). It has been reported that Ca2+ and Mg2+ can bind to enzymes to form a stable conformation and improve the catalytic effect (Oyekola et al. 2007).
Kinetic experiment of β-glucosidase puri ed from F. chlamydosporum HML278 By using pNPG as a substrate, it was shown that the Km and Vmax values of β-glucosidase were 2.76 mg/mL and 20.6 U/mg, respectively.

Discussion
This study reported for the rst time that F. chlamydosporum HML278 can utilize sugarcane bagasse as carbon source for solid-state fermentation to produce high-temperature-resistant β-glucosidase. By employing nondenaturing gel recovery and gel ltration chromatography, two β-glucosidase BG FH1 and BG FH2 were puri ed, with molecular weights of 93 kDa and 52 kDa, respectively. Puri ed BG FH1 and BG FH2 were β-glucosidase enzymes with high transglycosidic activity. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolytic activity and hydrolyzed cellobiose into glucose. At meantime, the enzymes had transglycosidic activity and can synthesize cellobiose and cellotriose using low molecular weight monosaccharides.
The optimum temperature for puri ed BG FH1 and BG FH2 from F. chlamydosporum HML278 was 60 ℃, and the enzymes were stable below 70 ℃. The enzymes had highest activity at pH 6.0, and were stable in the pH ranging from pH 4.0 to pH 10.0. Ag 2+ , Co 2+ , Cu 2+ , Zn 2+ , and Hg 2+ had strong inhibitory effect on the puri ed enzymes, while Mn 2+ , Ca 2+ , Mg 2+ , and Fe 3+ had obvious activation effect on the enzymes, and Zn 2+ and Ni + had no obvious effects on enzymes. In addition, some peptide sequences were also identi ed by tandem time-of-ight mass spectrometry, but there was no relative information on these peptides in the protein database.
Heat-stable enzymes have obvious advantages as catalysts. Because high temperature can promote the enzyme penetration and cell wall destruction during the process (Kwon et al., 1994), the hydrolysis effect is usually better.
Thermophilic fungi are now considered to be a promising source for producing thermostable cellulase that used for cellulose degradation, and can increase the sacchari cation rate (de Cassia Pereira et al., 2015). By using nuclear magnetic resonance analysis, Makropoulou et al. found that β-glucosidase from Fusarium oxysporum has transglycosidic activity. It can catalyze a variety of disaccharides to generate β-D-glucose through transglycosidation. Fusarium oxysporum can directly hydrolyze cellulose and synthesize monoalcohol ethanol, glycol glycol, and triol glycerol after sacchari cation. Because β-glucosidase has transglycosidic activity, monoethanol alcohol is preferentially synthesized (Makropoulou et al., 1998). The cellulase produced by Fusarium spp. is heat-resistant. It has been reported that Fusarium spp. can also produce enzymes involved in alcohol production, which can saccharify cellulose materials while convert the ve-or six-carbon sugars to alcohol (Brunner and Lichtenauer, 2007;Gómez-Gómez et al., 2001;Maheshwari et al., 2000;Royer and Moyer, 1995;Kumar et al., 1991;Singh and Kumar, 1991;Vaidy and Seeta, 1984;Woo and McCrae, 1977;Sampathnarayanan and Shanmugasundaram, 1970). In conclusion, we characterized two β-glucosidases with both hydrolytic and transglycoside activities from F. chlamydosporum HML278 fermentation, and these identi ed enzymes have great potential in industrial application, such as bioethanol, papermaking, feed, food, textile, detergent, and pharmaceutical industries (Xie et al., 2015;Kim et al., 2011;Alani et al., 2008;Maheshwari et al., 2000).
Fusarium chlamydosporum HML278 is a High Cellulase Producing Strain selected by us.   Figure 1 Non-denaturing gel electrophoresis analysis of enzymes puri ed from Fusarium chlamydosporum HML278.

Figure 2
Puri cation of enzymes by HiPrep 16/60 Sephacryl S-200 h\High Resolution chromatography. The rst protein peak was BG FH1, and the second peak was BG FH2.

Figure 3
Silver staining of SDS-PAGE for puri ed -glucosidases produced by F. chlamydosporum HML 278. A, BG FH1; B, BG FH2. 1 and M were original fermented solution and protein marker, respectively. 2 and 3 were puri ed BG FH1 and BG FH2, respectively.

Figure 8
The optimum pH and effect of pH on the activity and stability of the β-Glucosidase from F.chlamydosporum HML 278.