Characterization of a novel detergent-resistant type I pullulanase from Bacillus megaterium Y103 and its application in laundry detergent

Abstract This study aims to find a moderate pullulanase for detergent industry. The pulY103B gene (2217 bp) from Bacillus megaterium Y103 was cloned and expressed in Escherichia coli. PulY103B contained four conserved regions of glycoside hydrolase family (GH) 13 and the typical sequence of type I pullulanase. The optimal reaction conditions of PulY103B were pH 6.5 and 40 °C. In addition, it remained stable below 40 °C and over 80% of activity was retained at pH ranging from 6.0 to 8.5. The best substrate for the enzyme was pullulan. Furthermore, it exhibited activity toward wheat starch (36.5%) and soluble starch (33.4%) but had no activity toward amylose and glycogen. Maltotriose and maltohexaose were major pullulan hydrolysis products. Soluble starch and amylopectin were mainly hydrolyzed into maltotetraose. These results indicated that PulY103B is a novel type I pullulanase with transglycosylation activity via formation of α-1,4-glucosidic linkages. Moreover, PulY103B was strongly stimulated by nonionic detergents [viz, Tween 20 (10%), Tween 80 (1%), Triton X-100 (20%)] and commercial liquid detergents (3.0 g/L). Wash performance tests demonstrated that the mixture of PulY103B and detergent removed starch-based stains better than using detergent alone (p < 0.05). Therefore, this pullulanase has big potential as a detergent additive.


Introduction
Pullulanase (EC 3.2.1.41) is one of debranching enzymes which could cleave a-1,6-glycosidic linkages in pullulan and starch-type polysaccharides. According to substrate specificity and hydrolytic property, pullulanases are classified as five types viz, type I, type II, and pullulan hydrolases (types I, II, and III). [1] According to amino sequence, the type I and II pullulanases are categorized into GH13 and GH57, respectively, pullulan hydrolases of types I and III are grouped in GH13, and the type II pullulan hydrolases belong to GH49; pullulanases in GH13 usually contain four conserved regions (I-IV). [2] Type I pullulanases contain the consensus sequence YNWGYDP. [3] In general, pullulanases are used to catalyze hydrolysis reaction. However, several pullulanases have transglycosylation activity [1,[4][5][6] and few of them can form a-1,4-glucosidic linkage. [1,4] Pullulanases are widely applied in starch-based industries. [2,7] Numerous studies have explored thermo/acid pullulanases and improved their thermo/acid stability. [8][9][10][11] However, research on pullulanases should focus on one that can work at moderate temperatures in consideration of ecofriendliness and cost-effectiveness. [3,12] Pullulanases especially type I that can work under moderate conditions, are used in granular starch hydrolysis, oligosaccharide production, dishwashing and laundry detergents. [12][13][14] Furthermore, only alkali-tolerant, and detergent-resistant pullulanases are more fitted for dishwashing and detergent industry. [2,3,15,16] Among pullulanases reported that are suitable as detergent additives, only a few pullulanases can work under moderate conditions. [3,15] Moreover, none of moderate pullulanase has been used in laundry industry.
Bacillus megaterium Y103 can reportedly produce an amylopullulanase (PulY103A) with transglycosylation activity. [1] Moreover, B. megaterium produces a type I pullulanase (BmPul, 112 kDa) which has no transglycosylation activity. [17] Both BmPul and PulY103A are unstable in detergent solutions. In this study, a novel moderate recombinated pullulanase from B. megaterium Y103 was characterized and was tested as an additive in laundry detergents.
The protein concentration was quantified according to the Bradford method. [21] Functional characterization of PulY103B The optimal pH was investigated in 50 mM various buffers (viz, phosphate-citrate, pH 5.0-6.5; sodium phosphate, pH 6.0-7.5; MOPS, pH 6.0-8.0; and glycine-NaOH, pH 8.0-10.0) under 40 C. For pH stability determination, the purified PulY103B was kept in different buffers under 40 C for 30 min. Then the residual activities were checked by standard assay.
The optimal temperature for pullulanase activity of PulY103B was determined at temperature ranged from 30 C to 50 C at pH 6.5 (50 mM, MOPS buffer). For the temperature stability study, PulY103B was incubated at different temperature conditions for 30 min in MOPS buffer (50 mM, pH 6.5). Then the residual activities were measured after treatment.

Effect of chemicals and metal ions on PulY103B
To study the influence of chemicals and metal ions on PulY103B, enzyme activity was assayed in the existence of 2 and 5 mM metal ions and different concentrations of chemical reagents. The metal ions used were Ca 2þ , Mg 2þ , Zn 2þ , Co 2þ , Cu 2þ , and Fe 2þ . The chemical reagents tested were Triton X-100, Tween 80, Tween 20, sodium dodecyl sulfate (SDS), ethylenediamine tetraacetic acid (EDTA), urea, and b-mercaptoethanol (b-ME). The control was measured with the enzyme dialyzed by standard assay.

Substrate specificity and kinetic parameters of PulY103B
Substrate specificity of PulY103B was determined with different substrates under the method of activity assay. All substrates were tested at the same concentration (10 mg/mL) and under the assay condition (pH 6.5, 40 C, 10 min). The substrates were pullulan, potato starch, sweet potato starch, wheat starch, soluble starch, dextrin, glycogen, amylose, amylopectin, and a/b/c-cyclodextrin.
The kinetic parameters for pullulan were studied, the reaction condition was similar to the method of activity assay using different substrate concentrations and reaction time of 5 min. The parameters were calculated by "GraphPad Prism" program.

Wash performance of PulY103B
In order to evaluate the wash performance of PulY103B, white cotton fabric pieces (8 Â 8 cm 2 ) were stained according to the method of Chinese Standard GB/T 13174-2008 with some modification. Staining solution contains two parts, protein/grease solution and starch solution (20.0 g/L wheat starch solution), and mixed at rate of 1:1 (v/v). Protein/ grease solution (homogeneity) contains 10.0 g/L gum arabic, 1% (v/v) carbon ink, 60.0 g/L whole milk powder, 10.0 g/L whole juice of the egg (egg white/yolk: 3/2), and 5% (v/v) soybean oil. The stained fabric pieces were dried at 60 C for 4 h. The stained fabric pieces were randomly separated into seven groups. The control group (CK) samples were dipped in separate flasks (200 mL) containing 50 mL of tap water (the same as the other groups). The rest six groups were as follows: T1: 3.0 g/L detergent (one of four brands liquid laundry detergents, viz, Blue Moon, Ariel, Comfort, and OMO), T2: T1 þ 0.1 U/mL of PulY103B, T3: T1 þ 0.2 U/ mL of PulY103B, T4: T1 þ 0.3 U/mL of PulY103B, T5: T1 þ 0.4 U/mL of PulY103B, and T6: T1 þ 0.5 U/mL of PulY103B. The enzyme activity was defined as the method of activity assay. [1] The flasks were put in a rotary shaker (150 rpm, room temperature) for 30 min. The samples were rinsed with tap water after washing and air dried. The stain removal effect was detected by brightness meter (Pnshar, Hangzhou, China) and represented by the value of detergency (R) and rate of the value of detergency (P).
In the formula, F 1 and F 2 represent the whiteness of fabric piece before and after washing [values from 0 (black) to 100 (white)]. R o represents the value of detergency of detergent without enzyme, while R a represents the value of detergency of the detergent with enzyme.

Scanning electron microscopy
The scanning electron microscopy (SEM) of fabrics was determined according to the method of Long et al. [22]

Statistical analysis
The method by Chen et al. [18] was used for statistical analysis.
Homology analysis (Figure 1) revealed that PulY103B contains four conserved regions (I, II, III, and IV) of GH 13 and the typical region (region A) of type I pullulanase. The results indicated that the three amino acids Asp406, Glu438, and Asp526 are the catalytic sites of the enzyme.

Purification of PulY103B
PulY103B was purified by Ni-IDA column (Supplementary  Table S1). After purification, its specific activity increased by 6.8-fold with a yield of 54.0%. The result ( Figure 2) showed that PulY103B was purified to homogeneity with an Mw of $83.2 kDa (SDS-PAGE), corresponding to the calculated Mw (84.2 kDa).

Biochemical characterization of PulY103B
The optimal reaction conditions of PulY103B were pH 6.5 and 40 C, and over 70% of its maximum activity was detected at pH 6.0 and 30 C (Supplementary Figure S1A and C). The enzyme was stable below 40 C (Supplementary Figure S1D), and over 80% of its activity was retained when it was incubated at pH 6.0-8.5 (Supplementary Figure S1B).

Impact of various chemicals and metal ions on PulY103B
Among the cations tested, Zn 2þ , Co 2þ , and Cu 2þ strongly inactivated PulY103B. Although Ca 2þ , Mg 2þ , and Fe 2þ evidently enhanced the enzyme activity (Table 1), Tween 80 (1%) and urea (1%) stimulated the activity of PulY103B by about 100%. Furthermore, Tween 20 and Triton X-100  enhanced the activity of PulY103B by over 100% at 10% concentration, whereas Triton X-100 stimulated the activity of PulY103B by about 50% even at 20% concentration. Moreover, SDS (1%), b-ME (1%), and EDTA (10 mM) resulted in about a 60% decrease in the enzyme activity. In addition, four commercial detergents, namely, Blue Moon, Ariel, Comfort, and OMO, stimulated the activity of the enzyme by about 20% at the recommended concentration (3.0 g/L) (data not shown).
TLC results showed that pullulan was hydrolyzed into maltotriose and probably one of malto-oligosaccharides (DP ! 6) as the main products ( Figure 3A). Soluble starch and amylopectin were hydrolyzed into maltotetraose as the main product at the end, and a few byproducts were also produced ( Figure 3B and C). Furthermore, the pullulan hydrolysis products were verified via HPLC, which revealed that the enzyme mainly hydrolyzed pullulan into maltotriose and maltohexaose, meanwhile, trace of maltononaose was detected ( Figure 3D and E).

Wash performance of PulY103B
As shown in Table 2, the addition of PulY103B significantly improved the detergency of all detergents tested (p < 0.05). The maximum value of R reached 56.27 when 0.5 U/mL of PulY103B was added to 3.0 g/L of Blue Moon. Similarly, the value of P presented the same trend and obtained the  highest value (1.94) at the same condition of detergency. However, no significant differences were observed in the values of R and P between T5 and T6 for all detergents tested (p < 0.05). The results of stain removal test of PulY103B (Supplementary Figure S2) and the results of SEM (Supplementary Figure S3) suggested that the enzyme combined with the commercial detergent Blue Moon (3.0 g/L) at 25 C was remarkably better than the detergent alone in removing starch-based stains.

Discussion
A recombinated pullulanase, PulY103B belonging to the GH 13, was characterized and its application potential was checked. The Mw of PulY103B exhibits high identity to that of type I pullulanase from Bacillus methanolicus. [12] Furthermore, PulY103B has similar biochemical characteristics to moderate type I pullulanases from hotspring metagenome and white edible mushrooms. [23,24] Pullulanases from B. megaterium (BmPul, PulY103A, and PulY103B) have similar stability, but there are some differences in their reaction properties. [1,18] Only PulY103B probably has big potential for granular starch hydrolysis (around 35 C, pH 5-6) for showing high activity around 30 C and pH 6.0. [3,12] Moreover, over 80% of its activity could be retained at pH 6.0-8.5. Thus, PulY103B may be used as a detergent enzyme in liquid detergent working under moderate condition. Only alkali-stable, and detergent-resistant pullulanases from Bacillus pseudofirmus 703 and Exiguobacterium sp. SH3 could work under moderate condition as mentioned above. [3,15] However, their application potential as detergent additive have not been investigated.
PulY103B was strongly inhibited by Cu 2þ , Zn 2þ , and Co 2þ but activated by Ca 2þ and Mg 2þ , properties are similar to those of type I pullulanase from B. methanolicus (Pul M ). [24] However, PulY103B activity was enhanced by Fe 2þ , unlike Pul M but consistent with alkali-tolerant type I pullulanase from Exiguobacterium acetylicum. [25] EDTA and b-ME are inhibitors to the enzyme, indicating that the enzyme is a metalloenzyme and probably thiol group has effect on catalysis. The enzyme was the most stable pullulanase against nonionic detergents (viz, Tween 80, Tween 20, and Triton X-100) and denaturation agent of urea, which indicated that it has big potential for application in liquid detergents. [2,15,26] In view of the characteristics of PulY103B, its stability under commercial liquid detergents was tested. Result showed that PulY103B was stimulated by the four commercial liquid detergents tested herein (Blue Moon, Ariel, Comfort, and OMO) by about 20% at recommended concentration. To date, only amylopullulanase from Clavispora lusitaniae ABS7 has been reported to be stimulated by commercial detergents. [16] The substrate specificity of PulY103B was different from that of PulY103A and BmPul. It could not act on amylose and malto-oligosaccharides, suggesting that PulY103B is not an amylopullulanase. [1] Meanwhile, the enzyme had a broader substrate specificity than BmPul especially on several kinds of starch (BmPul could only act on pullulan and amylopectin). Although PulY103B has similar K m for pullulan as BmPul (0.33 mg/mL), it has bigger K cat for pullulan than BmPul (0.46 s À1 ), [17] which presented that PulY103B catalyzed pullulan hydrolysis more efficiently compared with BmPul. These results incorporating with the enzyme characterization showed that PulY103B could probably deal with starch-based stains as a detergent enzyme. Soluble starch and amylopectin were hydrolyzed into maltotetraose with a few byproducts by the enzyme, in contrast to other pullulanases that have been reported. And it may be useful in the production of maltotetraose. Furthermore, maltotriose and maltohexaose were the main products of pullulan hydrolysis, maltononaose was also detected, which was similar to PulY103A. Thus, the products of maltohexaose and maltononaose should be produced via the a-1,4-transferring activity of the enzyme. [1] As mentioned above, PulY103B is the first type I pullulanase that exhibits a-1,4-transferring activity.
Wash performance analysis revealed that the use of PulY103B as a detergent enzyme could significantly improve the detergency of commercial liquid detergents against starch-based stains (p < 0.05) under moderate condition, consistent with the characteristics of PulY103B. Among the commercial detergents tested herein, the treatment of Blue Moon (3.0 g/L) combined with the enzyme (0.5 U/mL) obtained the maximum values of R and P. Therefore, the enzyme was more adapted to Blue Moon than to the other commercial detergents. Furthermore, no significant differences were observed between T5 and T6 (p < 0.05), indicating that T5 (3.0 g/L detergent combined with 0.4 U/mL PulY103B) is the best solution for the removal of starch-based stains. Hence, the enzyme has a big potential as a detergent additive in liquid detergents working under moderate condition. Among the studies that evaluated the wash performance of pullulanases, [16,26] no one performed quantitative analysis. the method adopted herein was more reasonable.

Conclusion
The commercial hit of detergent enzymes is due to their special properties. Moderate reaction condition are the key to ensuring the environmental friendliness and sustainability of detergent enzymes. As detergent enzymes, moderately detergent-resistant type I pullulanase can address the high viscosity of starch solution at low temperature, In this study, a recombinated moderate type I pullulanase (PulY103B) was characterized. PulY103B is a moderate, alkali-tolerant, and detergent resistant pullulanase, and tests revealed that it has the typical characteristics of detergent enzymes. Wash performance experiments demonstrated that the enzyme has big potential as a detergent additive in liquid detergents.