Biochemical properties of a novel chitosanase from Bacillus amyloliquefaciens and its use in membrane reactor

Accepted Manuscript Biochemical properties of a novel chitosanase from Bacillus amyloliquefaciens and its use in membrane reactor Zhen Qin, Sa Luo, Yun Li, Qiming Chen, Yongjun Qiu, Liming Zhao, Lihua Jiang, Jiachun Zhou PII:

S0023-6438(18)30538-3

DOI:

10.1016/j.lwt.2018.06.027

Reference:

YFSTL 7211

To appear in:

LWT - Food Science and Technology

Received Date: 8 February 2018 Revised Date:

12 June 2018

Accepted Date: 13 June 2018

Please cite this article as: Qin, Z., Luo, S., Li, Y., Chen, Q., Qiu, Y., Zhao, L., Jiang, L., Zhou, J., Biochemical properties of a novel chitosanase from Bacillus amyloliquefaciens and its use in membrane reactor, LWT - Food Science and Technology (2018), doi: 10.1016/j.lwt.2018.06.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Biochemical properties of a novel chitosanase from Bacillus amyloliquefaciens

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and its use in membrane reactor

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Zhen Qinab, Sa Luoa, Yun Lia, Qiming Chenab, Yongjun Qiuab, Liming Zhaoab*, Lihua

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Jiangab, Jiachun Zhouab

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a

School of Biotechnology, State Key Laboratory of Bioreactor Engineering, R&D

Center of Separation and Extraction Technology in Fermentation Industry, East China

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University of Science and Technology, Shanghai 200237, China

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Shanghai Collaborative Innovation Center for Biomanufacturing Technology

(SCICBT), Shanghai 200237, China

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* Corresponding author.

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E-mail address: [email protected] (L.M. Zhao)

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Fax: +86 021-64250829

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No. 130 Meilong Road, Shanghai 200237, China

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Abstract Chitooligosaccharides are widely used in functional food, medicaments and

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agriculture. Controllable conversion of chitosan to chitooligosaccharides with desired

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degree of polymerization (DP) is of great value. Herein, a novel glycoside hydrolase

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family 46 chitosanase (BaCsn46A) was cloned and expressed from Bacillus

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amyloliquefaciens. BaCsn46A exhibited good enzymatic properties with high specific

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activity (1031.2 U/mg) under optimal hydrolysis conditions of pH 6.0 and 50°C.

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BaCsn46A is an endo-type chitosanase, which could be utilized to yield abundant

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chitooligosaccharides. Furthermore, the preparation of high degree of polymerization

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chitooligosaccharides (DP 5–10) was investigated by BaCsn46A in an enzymatic

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reactor with a subsequent series of membranes. The maximum concentration of total

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chitooligosaccharides (DP 2–10) was 28.11 g/L (93.70% yield). The proportion of high

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degree of polymerization chitooligosaccharides (DP 5–10) occupied 56.17% (w/w) of

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total chitooligosaccharides. This study provided a cleaner production process for the

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controllable

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polymerization.

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preparation of chitooligosaccharides

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with

specific

degree

of

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KEYWORDS: Chitosanase; Chitooligosaccharides; Glycoside hydrolase family 46;

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Enzyme membrane reactor; Product selectivity

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1. Introduction Chitooligosaccharides are the degradation products of chitosan, which showed

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various functional properties, including antimicrobial, antitumor, antioxidant,

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immunostimulatory and elicitors of plant defence (Aam et al., 2010). As beneficial

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biological activities, chitooligosaccharides are widely used in functional food,

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medicaments and agriculture (Das et al., 2015; Zou et al., 2016). Commercial

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chitooligosaccharides are often obtained by enzymatic hydrolysis of chitosan. The

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specific chitosan hydrolytic enzyme is chitosanase (E C 3.2.1.132). However, as the

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endo-type catalytic mode, chitosanases often randomly cleave β-1,4-glycosidic bond

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inside of chitosan, and release a mixture of uncontrollable chitooligosaccharides. Most

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of the reported chitosanases tend to hydrolyze chitosan to produce chitobiose,

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chitotriose and chitotetraose as final hydrolytic products rather than higher degree of

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polymerization (DP) chitooligosaccharides (Nidheesh, Pal & Suresh, 2015; Zhang, et

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al., 2015). The high DP chitooligosaccharides are intermediate products, which are

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continuously degraded in the reaction progress (Kuroiwa et al., 2009; Lin, Hsiao &

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Chiang, 2009). However, the biological activities of chitooligosaccharides depend on

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their DP (Sanchez et al., 2017; Sinha, Chand & Tripathi, 2016). Some previous studies

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confirmed that high DP chitooligosaccharides exhibited more significant physiological

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activity to be utilized in functional foods and pharmaceutical than the low DP

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chitooligosaccharides (Kuroiwa, et al., 2009; Li, Xing, Liu & Li, 2016; Lin et al.,

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2009). Therefore, obtaining novel specific chitosanase and investigating controllable

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bioconversion process are highly beneficial for the production of high value

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chitooligosaccharides in industrial scale. The

development

of

membrane

separation

technology

introduces

enzyme-membrane coupling process (also named enzyme membrane reactor) in which

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continuous reaction occurs simultaneously with separation of the target product from

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the reaction mixture (Pinelo, Jonsson & Meyer, 2009). Thus the enzyme-membrane

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coupling process has the potential to separate high DP chitooligosaccharides from the

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hydrolytic

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chitooligosaccharides being further hydrolyzed. Moreover, a membrane process after

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the enzymatic treatment allows the continuous separation the products from reaction

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mixture, which decrease product inhibition in the enzymatic reaction and promote the

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cyclic utilization of enzyme (Andric, Meyer, Jensen & Dam-Johansen, 2010). To date,

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most of the chitooligosaccharides enzyme-membrane coupling studies focus on the

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continuous production of chitooligosaccharides (Jeon & Kim, 2000; Kuroiwa, et al.,

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2009; Santos-Moriano, Woodley & Plou, 2016). Some previous studies indeed

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demonstrated that high DP chitooligosaccharides could be enriched through enzyme

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membrane bioreactor, the final yields of high DP chitooligosaccharides reached 46–69 %

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in total (Kuroiwa et al., 2009; Lin et al., 2009). However, the high investment of

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pressurized membrane reactor and the lack of suited low-cost specific chitosanases

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limits the further process engineering investigation of enzyme-membrane coupling

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technology.

and

prevent

the

intermediate

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mixture,

high

DP

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In this study, the gene cloning, expression and biochemical properties of a novel

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chitosanase from Bacillus amyloliquefaciens were investigated. Furthermore, the 4

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matching technique for producing high DP chitooligosaccharides was investigated by

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hydrolysis

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ultrafiltration-nanofiltration membrane system. This study provided potential

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enzyme-membrane coupling technology and relevant enzyme for the preparation of

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high DP chitooligosaccharides.

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2. Materials and methods

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2.1. Cloning, expression and enzyme purification

in

an

enzymatic

reactor

in

series

with

an

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chitosan

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of

B. amyloliquefaciens YX-01 used in this study was isolated from fermented fruits

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beverages samples. From the genome information of B. amyloliquefaciens (He et al.,

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2012), a putative GH family 46 chitosanase gene (GenBank: AFJ63438) was identified.

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To express this gene (BaCsn46A) in E. coli, the gene fragment was amplified from the

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genomic DNA of B. amyloliquefaciens YX01. NheI and XhoI sites (underlined) were

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added

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ATTCTAGCTAGCGCCGGGCTGAATAAGGATC;

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CCGCTCGAGTTATTGAATAGTGAAATTACCGTATTCGC), respectively. The

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PCR products were gel-purified, digested, and cloned into pET-28a vector, and the

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recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells for

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gene expression.

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The recombinant protein was concentrated and purified as described before (Qin,

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Yan, Yang & Jiang, 2016). Briefly, the cells were harvested and suspended in buffer A

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(50 mmol/L Tris–HCl pH 8.0, 500 mmol/L NaCl, 20 mmol/L imidazole), then

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disrupted by sonication. The debris was removed by centrifugation at 10,000×g for 5 5

ACCEPTED MANUSCRIPT min, and the supernatant was passed through a Ni-IDA column (1 × 5 cm) (GE Life

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Sciences, USA) pre-equilibrated with buffer A. The column was washed with 15

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column volumes (CV) of buffer A followed by 5 CV of buffer B (Buffer A containing

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50 mmol/L imidazole), and the recombinant protein was eluted by 5 CV of buffer C

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(Buffer A containing 200 mmol/L imidazole). The purified protein fractions were

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combined, concentrated and buffer exchanged in buffer (20 mmol/L Tris-HCl, 100

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mmol/L NaCl, pH 7.5) for subsequent experiments. Protein concentration was

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measured by the Bradford method Kit (Sangon, Shanghai, China), using BSA as the

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standard. Briefly, serial dilutions of the protein samples (100 µL, final concentrations

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of 0 to 200 µg/mL) were prepared. Then added 1.0 mL of coomassie blue reagent to

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each tube and mixed by vortex. After 5 minutes, the protein concentration was

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determined by measuring absorbance at 595 nm and calculated through the calibration

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curve (R2=0.9995). The association form and the molecular weight of BaCsn46A were

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estimated by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass

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Spectrometry (MALDI-TOF MS; AB SCIEX TOF/TOFTM 4800 System, AB SCIEX,

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USA).

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2.2. Sequencing and phylogenetic analysis

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N-terminal

signal

peptide

was

analyzed

using

SignalP

4.1

Server

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(http://www.cbs.dtu.dk/services/SignalP/). Database homology searches of sequences

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were carried out using BLAST in GenBank at the NCBI. Sequence analysis and

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multiple alignments were performed by Clustal W program and ESPript.

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2.3. Enzyme assay, substrate specificity and kinetic parameters 6

ACCEPTED MANUSCRIPT Chitosanase activity was assayed using 3,5-dinitrosalicylic acid (DNS) method as

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previous study (Qin et al., 2018). Namely, 50 µL properly diluted enzyme solution was

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added into 350 µL of 0.5 % (w/v) chitosan (degree of deacetylation ≥ 95%; molecular

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weight 150–300 kDa; Aladdin, Shanghai, China) substrate mixture (prepared in 50

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mmol/L sodium acetate buffer pH 6.0, which was preincubated at 50 °C for 5 min). The

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reaction mixture was incubated at 50 °C in a water bath for 10 min. The reaction was

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terminated by adding 600 µL DNS reagent and boiled for 10 min. The reducing sugar in

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the supernatant was determined by measuring absorbance at 540 nm, using

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D-glucosamine as standards. The dilution ratio of enzyme solution was based on the

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absorbance of final mixture at 540 nm, namely the absorbance values should be ensured

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within 0.2-0.8. The reactions were done in triplicate and their mean and standard

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deviation values were used for analysis. One unit (U) of chitosanase activity was

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defined as the amount of enzyme liberating 1 µmol D-glucosamine-equivalent reducing

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sugars per minute under the above conditions.

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Substrate specificity of BaCsn46A was determined by measuring the enzyme’s

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activity in the presence of different substrates such as chitosan, colloidal chitin and

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microcrystalline cellulose. The enzyme activity was determined under standard

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conditions. The kinetic parameters were analyzed using various concentrations of

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chitosan, ranging from 0.5 to 10.0 mg/mL. The reactions were performed at 50 °C for 5

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min in 50 mmol/L sodium acetate buffer pH 6.0. Experiments were carried out in

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triplicate. By plotting reaction rate against concentration, Km and Vmax values were

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calculated using the nonlinear regression analysis of the Michaelis–Menten equation by

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program Grafit.

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2.4. Characterization of BaCsn46A Chitosan was used as substrate to determine the enzymatic characterization of

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BaCsn46A.The optimal pH for purified BaCsn46A was determined by measuring the

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activity from pH 4.5 to 8.0 using 50 mmol/L various buffers. To determine pH

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stability, residual activity was measured after incubation of the enzyme at 20 °C for

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30 min in the 50 mmol/L various buffers. The optimal temperature was determined at

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30–70 °C in 50 mmol/L sodium acetate buffer pH 6.0. Enzymatic activity was

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determined under standard conditions. Thermostability of the enzyme was determined

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by measuring residual activity after incubation of the enzyme at different

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temperatures (30–70 °C) for 30 min in 50 mmol/L sodium acetate buffer pH 6.0.

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Thermostability means the quality of enzyme to resist irreversible inactivation at a

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high relative temperature. The enzyme activity was determined by standard enzyme

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assay conditions mentioned above. The concentration of chitosan substrate was 0.5 %

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(w/v) and the concentration of enzyme solution (about 0.05-0.5 µg/mL) was diluted

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based on the absorbance values mentioned above.

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2.5. Hydrolytic pattern of BaCsn46A

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The hydrolytic properties of BaCsn46A were investigated by analyzing the

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hydrolysis products from the substrates including chitosan and chitooligosaccharides

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(chitobiose: (GlcN)2; chitotriose: (GlcN)3; chitotetraose: (GlcN)4; chitopentaose:

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(GlcN)5 and chitohexaose: (GlcN)6) (purity ≥ 95%, Long Dragon Bio., Huizhou,

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China). BaCsn46A (1.5 U/mL) was added to 1% (w/v) chitosan and then incubated at 8

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50 °C for 2 h in 50 mmol/L acetate buffer pH 6.0 (reaction volume: 500 µL). For the

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chitooligosaccharides,

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chitooligosaccharides, and then incubated at 50 °C for a certain reaction time in 50

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mmol/L acetate buffer pH 6.0 (reaction volume: 100 µL). Samples withdrawn at

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different times were immediately boiled for 5 min, and then analyzed by Thin-Layer

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Chromatography (TLC) method. Samples were spotted on a TLC plate (Silica gel 60

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F254, Merck, Darmstadt, Germany), developed in isopropanol: water: ammonium

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hydroxide (15:1:7.5, v/v) as solvent, and sprayed with alcohol: p-anisaldehyde:

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sulfuric acid: acetic acid (89:5:5:1, v/v). The hydrolysis products were visualized by

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heating the plate at 130 °C in an oven for a few minutes.

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2.6. Enzyme-membrane coupling reactor system

U/mL)

was

added

to

1%

(w/v)

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(1.5

A spiral-wound membrane system (Ruina membrane engineering, Hangzhou, China)

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BaCsn46A

was coupled with a 5 L stainless steel jacket reaction tank to build the

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enzyme-membrane coupling reactor system (Fig. 1). The membrane was a

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polyethersulfone UF membrane with molecular weight cut-off 5000 Da (Risingsun,

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Beijing, China). The effective membrane area was 0.37 m2. The impeller pump

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installed in the spiral-wound membrane system was used to provide transmembrane

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pressure and link the membrane module and the reaction tank to circulate reaction

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solution through the system. Warm water from a circulating water bath flowed

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continuously through the jacket of reaction tank to maintain a constant reaction

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temperature.

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For the substrate preparation, chitosan powder was dissolved in 0.05 mol/L 9

ACCEPTED MANUSCRIPT acetate buffer at pH 5.5 to obtain a 30 g/L of chitosan solution. The homogeneous

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chitosan solution (3 L) was further moved to the reaction tank, and the catalytic

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reaction was started by adding 100 U/g crude enzyme of BaCsn46A. The reaction

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mixture was maintained at 40 °C for 150 min. In order to reduce the viscosity of the

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chitosan solution, the enzymatic reaction was first proceeding for 20 min in the

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reaction tank. Then the pump was turned on to pump the mixture in the reaction tank

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through the membrane module with 5 bar pressure. The permeate was diverted to the

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product tank of tandem nanofiltration system, and the retentate was returned circularly

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to the reaction tank for further reaction. The proper amount of 0.05 mol/L acetate

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buffer at pH 5.5 was continuously fed to the reaction tank from another feed tank to

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maintain a minimum volume of the mixture in the reaction tank. At various time

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intervals, samples of permeate and retentate were withdrawn at different times and

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immediately boiled for 5 min. The concentrations of different chitooligosaccharides

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were analyzed by TLC and High Performance Liquid Chromatography (HPLC). The

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content of total chitooligosaccharides was calculated by the total concentration of

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different chitooligosaccharides in permeate from the product tank multiply by permeate

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volume. The yield was calculated by following formula:

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Yield =

C×V M

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C: the total concentration of chitooligosaccharides;

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V: the volume of permeate from the product tank;

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M: the content of initial chitosan.

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2.7. Nanofiltration concentration of chitooligosaccharides products 10

ACCEPTED MANUSCRIPT In order to concentrate the chitooligosaccharides products, desalinate reaction

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mixture and recycle the acetate buffer from aforementioned enzyme-membrane

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coupling reactor system, a nanofiltration system was used in series (Fig. 1). A

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spiral-wound NF membrane system (Ruina membrane engineering, Hangzhou, China)

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was coupled with aforementioned enzyme-membrane coupling reactor system. The

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memberane was a three-layer thin-film composite NF membrane with molecular

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weight cut-off 300 Da (Risingsun, Beijing, China). The effective membrane area was

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0.37 m2. After the volume of feed liquid in the product tank was more than the dead

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volume of nanofiltration system, the pump was turned on to pump the feed liquid

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through the nanofiltration membrane module with 10 bar pressure. The permeate was

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diverted to the reaction tank of aforementioned enzyme-membrane coupling reactor

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system, and the retentate was returned to the product tank of nanofiltration system

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(Fig. 1). After 150 min, the previous step of ultrafiltration system was turned off and 6

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L deionized water was gradually added into the product tank. The nanofiltration

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system continued working until the feed liquid was concentrated about 3 L. The

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concentrations of different chitooligosaccharides in the final products were analyzed by

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MALDI-TOF MS and HPLC. The yield of total chitooligosaccharides was calculated

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by above formula.

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2.8. Hydrolysis of chitosan by free BaCsn46A

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In order to compare the differences between the enzyme-membrane coupling process

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and uncontrolled enzymatic hydrolysis process during the chitooligosaccharides

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preparation. Enzymatic reaction in bulk system with same conditions was further 11

ACCEPTED MANUSCRIPT investigated. The hydrolytic process was carried out under the following conditions:

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pH 5.5, 40 °C, substrate concentration 3%, enzyme concentration 100 U/g, reaction

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time 150 min, stirring rate 80 rpm/min, reaction volume 3 L. Samples were withdrawn

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at different times (the same as enzyme-membrane coupling process) and immediately

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boiled for 5 min, followed by TLC and HPLC analysis.

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2.9. Chitooligosaccharides analysis methods

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The chitooligosaccharides mixtures were analyzed by HPLC system (Shimadzu

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20A, Shimadzu, Kyoto, Japan) equipped with evaporative light-scattering detector

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(ELSD). The chitooligosaccharides were separated on high-performance sugar

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column (Shodex Asahipak NH2 P-50 4E, Shodex, Kyoto, Japan), eluted by acetonitrile

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and distilled water (70/30) mixture with a flow rate of 1.0 mL/min at 30 °C. The

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concentrations of different chitooligosaccharides were quantified by integrating peak

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areas based on the respective standard curve. The pure standards of high DP

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chitooligosaccharides (DP>6) were unable purchased or obtained, therefore, the high

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DP chitooligosaccharides were identified by the sequence of peaks appeared in the

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HPLC chromatogram, and the concentrations of these chitooligosaccharides were

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estimated based on the standard curve of chitohexaose (Lin et al., 2009). In order to

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determine the DP of chitooligosaccharides, mixtures were analyzed by MALDI-TOF

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MS.

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3. Results

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3.1. Sequence analysis, expression and purification of BaCsn46A

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The full-length BaCsn46A gene had an open reading frame (ORF) of 843 bp 12

ACCEPTED MANUSCRIPT encoding 280 amino acids with a theoretical molecular mass of 31.62 kDa and pI of

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8.83. A signal peptide of 38 amino acids was predicted by SignalP analysis. The active

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sites were highly conversed in all reported GH family 46 enzymes (Supplementary Fig.

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1). Two strictly reserved residues, Glu57 and Asp73, were hypothesized to be the

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general acid/base and the nucleophilic catalytic residues in BaCsn46A, respectively.

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To study the enzymatic properties of this GH family 46 enzyme without potential

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interference from the signal peptide, only the nucleotide sequence corresponding to the

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GH family 46 catalytic module was cloned into the vector pET28A. BaCsn46A was

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purified by one step of affinity chromatography with a purification factor of 5.86 and a

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recovery yield of 53.5% (Supplementary Table S1). The recombinant BaCsn46A

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migrated as a single band with molecular mass of 31 kDa on SDS-PAGE (Fig. 2A), in

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good accordance with the predicted molecular weight. MALDI-TOF MS analysis

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showed that BaCsn46A are mainly present as monomers in the solution and the

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monomeric molecular weight of BaCsn46A was about 29.68 kDa (Fig. 2B).

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3.2. Biochemical properties of BaCsn46A

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Effects of pH and temperature on the chitosanase activity of BaCsn46A were

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examined. BaCsn46A was most active at pH 6.0 in acetate buffer (Fig. 3A) and

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exhibited better activity within pH 4.0–9.5 as more than 80% of its activity was

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retained after incubation in various buffers for 30 min (Fig. 3B). It is noteworthy that

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BaCsn46A was unstable in McIlvaine buffer, which showed about half activity

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towards optimal buffer (acetate buffer) and partly inactivated after incubation in

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McIlvaine buffers (pH 3.0–5.0) for 30 min. The optimal temperature for BaCsn46A

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ACCEPTED MANUSCRIPT was determined to be 50 °C, and retained over 90% of its activity between 45 °C and

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60 °C (Fig. 3C). The suitable catalytic temperature of BaCsn46A for application was

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below 45 °C. It retained more than 80% of its initial activity after incubation at 45 °C

289

for 30 min (Fig. 3D).

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BaCsn46A showed relative high specific activity towards chitosan (1031.2 U/mg),

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but exhibited no activity towards other β-1,4 linked polysaccharide such as colloidal

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chitin and microcrystalline cellulose. The kinetic parameters, Vmax and Km of

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BaCsn46A towards chitosan were determined to be 7142.9 µmol/min.mg and 2.8

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mg/mL.

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3.3. Hydrolysis pattern of BaCsn46A

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The hydrolysis of chitosan by the purified BaCsn46A was performed. The DP of

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oligosaccharides products from BaCsn46A was related to the reaction time. The

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degradation of chitosan produced a mixture of chitooligosaccharides (mainly DP 2–6)

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during the initial hydrolysis, with chitobiose and chitotriose as the end products (Fig. 4).

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To understand the details of the hydrolysis mechanism, hydrolysis patterns of various

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linear chitooligosaccharides were further investigated by TLC (Fig. 5). The enzyme

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hardly hydrolyzed chitobiose and chitotriose. However, it could effectively hydrolyze

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chitotetraose, chitopentaose and chitohexaose. Chitotetraose was gradually hydrolyzed

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to produce mainly chitobiose in 30 min (Fig. 5), whereas chitopentaose and

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chitohexaose was completely hydrolyzed to yield mainly chitobiose and chitotriose in 2

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h (Fig. 5).

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3.4. Hydrolysis of chitosan in the membrane reactor

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ACCEPTED MANUSCRIPT In consideration of the stability of BaCsn46A and the temperature tolerance of

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membrane material, the membrane reactor system worked at an operating temperature

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of 40 °C. The results show that the series of chitooligosaccharides produced was mainly

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DP2 to DP10 (Fig. 6A). Chitopentaose was the most abundant component in the

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reaction mixture. In the enzyme-membrane coupling reaction process, the entire

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chitooligosaccharides component gradually accumulated with reaction time (Fig. 6B).

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The concentration of high DP chitooligosaccharides (DP5–10, intermediate products)

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was not decreased during enzyme-membrane coupling reaction. The proportions of

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different chitooligosaccharides components were almost unchanged during the whole

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reaction process (Fig. 6C). After 150 min reaction, there was nearly no

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chitooligosaccharides and reducing sugar in the real-time permeate (Supplementary

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Fig. S2). The yield of total chitooligosaccharides (DP2–10) was 100.06 % by HPLC

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analysis. The part of high DP chitooligosaccharides (DP5–10) was 50.50 g, which

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occupied 56.08% (w/w) of total detected chitooligosaccharides.

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3.5. Nanofiltration of chitooligosaccharides products

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and

MALDI-TOF

MS

analysis

indicated

that

the

final

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chitooligosaccharides products after nanofiltration concentration mainly contained

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(GlcN)2 to (GlcN)10, no glucosamine was deteted in the final products (Fig. 7). After

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nanofiltration, the maximum concentration of total chitooligosaccharides (DP2–10)

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was 28.11 g/L, corresponding to 93.70% of the initial chitosan concentration. The ratio

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(w/w) of (GlcN)2, (GlcN)3, (GlcN)4, (GlcN)5, (GlcN)6, (GlcN)7–10 were 6.55%,

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16.51%, 20.77%, 28.51%, 11.56% and 16.10% respectively. The proportion of high 15

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DP

chitooligosaccharides

(DP5–10)

occupied

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chitooligosaccharides.

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3.6. Hydrolysis of chitosan by free BaCsn46A

56.17%

(w/w)

of

total

In order to evaluate the hydrolytic result of chitosan by enzyme-membrane coupling

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system, the time course of different chitooligosaccharides concentrations during the

335

batch hydrolysis of chitosan in the same condition using free BaCsn46A was

336

investigated. Free BaCsn46A could efficiently degrade chitosan, releasing a series of

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chitooligosaccharides (DP2–10) (Fig. 6D). The concentrations of the target high DP

338

chitooligosaccharides (DP5–10) increased in the initial reaction time (0–30 min) and

339

then decreased in the remaining reaction process (Fig. 6E). The hydrolysis of chitosan

340

yielded predominantly (GlcN)2–5 as the main end products (Fig. 6F). The final yield of

341

total chitooligosaccharides (DP2–10) was 86.85% during 150 min hydrolytic reaction.

342

The concentration of high DP chitooligosaccharides (DP5–10) was 9.90 g/L, which

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occupied 38.01% (w/w) of total chitooligosaccharides.

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4. Discussion

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Most chitosanases have been reported to display strict substrate specificity (Weikert,

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Niehues, Cord-Landwehr, Hellmann & Moerschbacher, 2017). In this study,

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BaCsn46A exhibited relatively high specific activity towards chitosan. No activity

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towards chitin and cellulose had been observed for BaCsn46A. A comparison of the

349

hydrolysis behavior of BaCsn46A to other GH family 46 chitosanases indicated that

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BaCsn46A produce (GlcN)2 and (GlcN)3 as the major end products, no glucosamine

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was detected during hydrolytic process. Moreover, BaCsn46A hardly hydrolyzed

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action pattern with a demand of at least four glucosamine residues for effective

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cleavage. The -2, -1, +1 and +2 subsites were necessary for the catalytic reaction of

355

BaCsn46A in the total six subsites of catalytic groove (Supplementary Fig. S3). Thus,

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the minimum substrate of BaCsn46A was (GlcN)4, and no GlcN yield in the

357

hydrolytic reaction.

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Biochemical properties analysis suggested that BaCsn46A was a typical endo-type

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GH family 46 chitosanase. The high specific activity and appropriate catalytic property

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of BaCsn46A suggested its potential use in conversion of chitosan into

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chitooligosaccharides with commercial value. There are many previous studies

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identified various valuable chitosanases (Thadathil & Velappan, 2014), however only

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a few subsequent studies investigated how to well use these valuable chitosanases and

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develop suitable chitooligosaccharides production process. Controllable enzymatic

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preparing chitooligosaccharides with desired DP are still complicated in industrial

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scale. As the endo-type catalytic mode of BaCsn46A, chitooligosaccharides with high

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DP (DP 5–10) were suitable substrates of BaCsn46A, which were hydrolyzed to small

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chitooligosaccharides during the subsequent hydrolytic process. If the target

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intermediate chitooligosaccharides products could be separated from the hydrolytic

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mixture containing the chitosanase, further hydrolyzing could be avoided and the

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high-value chitooligosaccharides with high DP could be obtained in higher yield.

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Therefore, the membrane separation technique was coupled with the hydrolytic process

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of BaCsn46A to produce the target intermediate chitooligosaccharides in the further

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studies. The molecular weight of BaCsn46A was about 30,000 Da and the initial average

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molecular weight of undegraded chitosan was about 150–300 kDa. In contrast, the

377

target intermediate chitooligosaccharides (DP 5–10) was between about 823 Da and

378

1628 Da. Thus the ultrafiltration membranes with the average pore sizes of 5000 Da

379

were choose in the in the following experiments. The target intermediate high DP

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chitooligosaccharides products could be separated from reaction mixture, and

381

BaCsn46A could be retained and recycled to reactor tank. Through a series membrane

382

separation system, the enzymatic hydrolysis of chitosan was performed with batch

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operation of enzyme-membrane reactor coupled with nanofiltration membrane. After

384

nanofiltration, the maximum total concentration of chitooligosaccharides (DP2–10)

385

was 28.11 g/L, corresponding to 93.70% of the initial chitosan concentration. There

386

were nearly no acetate detected form HPLC analysis in the chitooligosaccharides

387

product (Fig. 7A), which indicated that chitooligosaccharide syrup was effectively

388

concentrated and desalinated by single step nanofiltration. Furthermore, the

389

spiral-wound membrane reactor could avoid membrane fouling in the hydrolytic

390

process comparing with previous flat sheet enzyme-membrane reactor (Kuroiwa, et al.,

391

2009). More importantly, the proportion of high DP chitooligosaccharides (DP5–10)

392

occupied 56.17% (w/w) of total chitooligosaccharides. The yield of target high DP

393

chitooligosaccharides (DP5–10) was higher than previous immobilized enzyme

394

bioreactor, membrane bioreactor and free chitosanase hydrolysis studies (Kuroiwa, et

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al., 2009; Ming, Kuroiwa, Ichikawa, Sato & Mukataka, 2006; Nidheesh et al., 2015).

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the target high DP chitooligosaccharides initially increased with time and then

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decreased after peaking under the same reaction condition (Fig. 6E). These

399

oligosaccharides were intermediate products of the hydrolysis reaction and were

400

degraded by further enzymatic reaction. Moreover, chitooligosaccharides products in

401

high

402

chitooligosaccharides products increased slowly after 70 min incubation. The final

403

yield of total chitooligosaccharides by free BaCsn46A (86.85%) was lower than

404

enzyme-membrane coupling system (93.70%) during the same reaction time. Thus it is

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hardly to obtain abundant high DP chitooligosaccharides by free BaCsn46A in high

406

yield. Without enzyme-membrane coupling operation, low DP chitooligosaccharides

407

(DP2–4) would gradually be primary hydrolytic products by free BaCsn46A. These

408

results illustrated that efficient preparation of the intermediate chitooligosaccharides

409

products required that the enzymatic hydrolytic progress of chitosan should be

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appropriately controlled.

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5. Conclusion

chitosanase's

reaction

rate.

The

yield

of

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concentration

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In conclusion, a novel chitosanase (BaCsn46A) from B. amyloliquefaciens was gene

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cloned, expressed, and biochemically characterized. BaCsn46A is a monomer with a

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molecular mass of 29.68 kDa. It was most active at pH 6.0 and 50 °C. BaCsn46A

415

showed relative high specific activity towards chitosan (1031.2 U/mg) to yield a series

416

of chitooligosaccharides by an endo-type cleavage pattern. In addition, this study

417

demonstrated that hydrolysis of chitosan by BaCsn46A through an enzyme-membrane 19

ACCEPTED MANUSCRIPT reactor coupled with nanofiltration system was a viable and efficient method for the

419

preparation of high degree of polymerization chitooligosaccharides. The maximum

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total yield of chitooligosaccharides (DP 2–10) was 93.70%, and the proportion of high

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degree of polymerization chitooligosaccharides (DP 5–10) occupied 56.17% (w/w) of

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total chitooligosaccharides.

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Acknowledgements

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This work was financially supported by the National Natural Science Foundation of

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China (31701537), China Postdoctoral Science Foundation (2016M601530),

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Shanghai Sailing Program (17YF1403500), Fundamental Research Funds for the

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Central Universities (222201814031) and “Shu Guang” project of Shanghai

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Municipal Education Commission and Shanghai Education Development Foundation

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(15SG28).

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Characterization of a new family 75 chitosanase from Aspergillus sp. W-2.

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Figure legends

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Figure 1 Schematic diagram of the enzyme-membrane coupling reactor system.

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Figure 2 Protein analysis of BaCsn46A. (A) SDS-PAGE of proteins during

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purification of the recombinant BaCsn46A by Ni-IDA. Lane M: standard protein

501

molecular weight markers; Lane 1: supernatant of lysate cells; Lane 2: purified enzyme.

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(B) MALDI-TOF MS showing the association form and molecular weight of

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BaCsn46A.

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Figure 3 The effects of temperature and pH on activity of the purified BaCsn46A. (A)

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Optimum pH, acetate buffer (■), McIlvaine buffer (●), phosphate buffer (▲); (B) pH

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(▼), glycine-NaOH buffer (♦); (C) Optimum temperature and (D) Thermostability. For

508

thermostability, residual activity was measured after incubating the enzyme at different

509

temperatures for 30 min in 50 mM acetate buffer (pH 6.0). The experiments were done

510

in triplicate and their mean and standard deviation values were used for analysis.

511

Figure 4 Hydrolytic process of BaCsn46A towards chitosan. M, marker sugars. The

512

reactions were performed at 50 °C in 50 mmol/L citrate buffer (pH 6.0) and analyzed

513

by TLC. (GlcN)–(GlcN)6: glucosamine, chitobiose, chitotriose, chitotetraose,

514

chitopentaose and chitohexaose, respectively.

515

Figure 5 Hydrolytic process of BaCsn46A towards chitooligosaccharides. M, marker

516

sugars. The substrates were (GlcN)2, (GlcN)3, (GlcN)4, (GlcN)5 and (GlcN)6,

517

respectively. The reactions were performed at 50 °C in 50 mmol/L citrate buffer (pH

518

6.0) and analyzed by TLC.

519

Figure 6 Enzymatic reaction pattern in the membrane reactor system (A–C) and free

520

enzyme system (D–F). (A) Total chitooligosaccharides of permeate by TLC analysis;

521

(B) Total chitooligosaccharides of permeate by HPLC analysis, glucosamine (■),

522

(GlcN)2 (●), (GlcN)3 (▲), (GlcN)4 (▼), (GlcN)5 (♦), (GlcN)6 (◄), (GlcN)7–10 (►)

523

and yield (○); (C) Composing proportion of different chitooligosaccharides monomers

524

in the permeate; (D) Total chitooligosaccharides of free enzymatic reaction mixture

525

by TLC analysis; (E) Total chitooligosaccharides of free enzymatic reaction mixture

526

by HPLC analysis; (F) Composing proportion of different chitooligosaccharides

527

monomers in the free enzymatic reaction mixture. The dot lines in each figures means

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Figure 7 HPLC analysis (A) and MALDI-TOF MS analysis (B) of final products by

530

enzyme-membrane coupling reactor system. The peaks in the spectra correspond to the

531

monoisotopic masses of sodium adducts [M+Na]+ of the chitooligosaccharides. The

532

final products contain chitooligosaccharides with DP 2–10.

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A high-activity chitosanase from B.amyloliquefaciens (BaCsn46A) was identified. BaCsn46A could be used for controllable producing chitooligosaccharides in membrane reactor. High degree polymerized chitooligosaccharides was accumulated by enzyme-membrane coupling process. This study provided a cleaner production process for controllable producing chitooligosaccharides.