Recyclable soluble-insoluble UCST-type PMAAc ... - ACS Publications

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Recyclable soluble-insoluble UCST-type PMAAc- cellulase biocatalyst for hydrolysis of cellulose into glucose Juan Han, Jing Wan, Yun Wang, Lei Wang, Chunmei Li, Yanli Mao, and Liang Ni ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00769 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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ACS Sustainable Chemistry & Engineering

Recyclable PMAAc-cellulase

soluble-insoluble biocatalyst

for

UCST-type hydrolysis

of

cellulose into glucose Juan Hana, Jing Wanb, Yun Wangb,*, Lei Wangb, Chunmei Lib, Yanli Maoc, Liang Nib a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013,

China b

School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang

212013, China c

Henan Key Laboratory of Water Pollution Control and Rehabilitation Technology,

Henan University of Urban Construction, Pingdingshan 467036, Henan, China *

School of Chemistry and Chemical Engineering, Jiangsu University, No.301, Xuefu

Road, Zhenjiang 212013, China. E-mail: [email protected]

ABSTRACT: How to improve the accessibility of immobilized cellulase to insoluble cellulose and recover immobilized enzyme from remaining insoluble substrate is a challenge to the efficient hydrolysis of cellulose into glucose. The objective of this present work is to solve the problems mentioned above by the immobilization of cellulase onto poly (methacrylamide-co-acrylic acid) (PMAAc), developing a reversibly soluble-insoluble biocatalyst with upper critical solution temperature (UCST) of 16 ºC. The as-prepared PMAAc-cellulase with a new UCST of 19 ºC exhibited significantly improved pH, temperature, storage and operation stabilities compared with that of free one, and about 82.4% of its original activity was retained even after ten cycles. Cellulase systems containing endo-β-1, 4-glucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (β-G) are co-immobilized at an optimum ratio on PMAAc by adjusting the additive amount of β-G, which can obtain higher hydrolysis efficiency. It was found that the co-immobilization of cellulase and β-glucosidase at the optimum ratio of 2.5:1 (w/w) showed excellent performance for the hydrolysis of cellulose and the yield of glucose was up to 89.1% at 50 ºC (>UCST) after 24 h, which was 58.4% and 15.4% higher than that of PMAAc-cellulase and free 1

ACS Paragon Plus Environment

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cellulase&β-glucosidase

respectively.

The co-immobilized

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PMAAc-cellulase&

β-glucosidase was still retained 61.48% of its original productivity after eight cycles of hydrolysis. This novel UCST-type polymer-enzyme catalytic system displays great potential in cellulose biorefinery. KEYWORDS: PMAAc, cellulase, biocatalyst, β-glucosidase, co-immobilized enzymes, hydrolysis

INTRODUCTION Cellulose, one of the most abundant renewable resources on the earth, can be converted into glucose by a chemical or enzymatic process. Then glucose can be further fermented to ethanol, which has been caused wide attention as a new green source of fuels for the future.1-3 It is generally recognized that cellulose can be specifically hydrolyzed into glucose by a complex cellulase system which mainly contains three classes of enzyme, endo-β-1,4-glucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (β-G).4 First, EG acts upon amorphous regions of cellulose chains to reduce the crystallinity and polymerization degree of cellulose, then CBH degrades crystalline cellulose from chain end to cut off cellobiose, which is further hydrolyzed into target product glucose by β-G.5 The compound cellulases have enormous biotechnological potential prospects in various industries, including textile, food fermentation, papermaking, and wastewater treatment, etc. However, native cellulase is hard to be recycled and easily deactivated during long-time operation, which has become the major limiting factors for biocatalyst in industrial application. Nowadays, many tools exist to stabilize biocatalysts, consisting mostly of immobilization, medium engineering, and protein engineering.6 Immobilization technology is widely applied to enzyme stability due to the significant characteristics of fast separation and recycling, resulting in the reduction of production cost. 7, 8 Insoluble magnetic nanoparticles (MNPs) bearing functional groups have been widely used as scaffolds for enzyme immobilization by physical adsorption or covalent coupling method.9,10 The weak physical adsorption between carriers and enzymes has the inherent flaw of continuous leakage in the catalytic process, and it is 2


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even hard to keep the enzyme fixed to the carrier under high standard industrial conditions.11,12 The carriers of adsorption usually are solid materials, which is difficult to separate from insoluble substrates.13,14 Therefore, the later method is a better choice in terms of stability because covalent interaction can inhibit spontaneous desorption of enzyme form support. However, the immobilization of enzyme onto solid MNPs suffers from low bioconversion efficiency compared with free enzyme due to diffusion-controlled mass transfer and steric hindrance with water-insoluble substrates.15,16 As for insoluble crystalline cellulose, the access of enzyme to substrate is potentially impeded and it is difficult to recover enzyme from remaining insoluble cellulose after hydrolysis reaction.17 The development of reversibly soluble-insoluble cellulase-polymer biocatalyst can solve the above mentioned problems. Stimuli-responsive polymers are a special class of polymer that can undergo soluble-insoluble change in response to external stimuli, such as temperature, pH and light, providing an alternative to enzyme immobilization.18 The immobilization of cellulase on stimuli-responsive carriers can achieve hydrolysis of insoluble cellulose and separation with residual insoluble substrate in a soluble state, and can further achieve recovery from resultant reaction solution in an insoluble state by simple temperature regulation. To data there are some reports using pH-sensitive polymer such as acrylate19 and succinyl chitosan20 as carriers, but the multistep pH-adjustment operation and limited pH range always lead to a relatively low activity yield. Another development of soluble-insoluble carrier is thermo-sensitive polymer such as poly (N-isopropylacrylamide) (PNIPAM) with lower critical solution temperature (LCST).21,22 However, in order to ensure that the thermo-sensitive polymer-enzyme catalyst exits in a soluble state, the hydrolysis of cellulose is required to carry out at a temperature below LCST which is not an optimum condition for hydrolysis reaction, and the recovery of biocatalyst was achieved by increasing temperature, resulting in the reduced enzyme activity and stability.23,24 A better alternative is developing a thermo-sensitive polymer with upper critical solution temperature (UCST) to produce UCST-type cellulase-polymer biocatalyst, which can exist in a soluble state and provide high accessibility to the 3



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substrate at a temperature above UCST and undergo precipitation below UCST, achieving high catalytic activity and easy recovery. At present, there are few researches on the immobilization of cellulase onto UCST-copolymer.25,26 Herein, a UCST-type polymer was synthesized through co-polymerization of methacrylamide (MAA) and acrylic acid (AAc) while the covalent immobilization of cellulase was realized after pre-activation of PMAAc with glutaraldehyde, and the biocatalyst was then used in a continuous system for hydrolysis of insoluble cellulose into glucose with high activity and recyclability. In order to obtain higher hydrolysis efficiency, cellulase systems containing EG, CBH and β-G were co-immobilized at an optimum ratio on PMAAc by adjusting the additive amount of β-G. The UCST-type PMAAc-cellulase catalysis system showed its obvious advantages in hydrolyzing insoluble cellulose in a soluble state at a relatively high temperature, which is superior to LCST-type polymer-enzyme system and MNP-enzyme system. EXPERIMENTAL SECTION Materials. Methacrylamide (MAA) and acrylic acid (AAc) were purchased from Aldrich (Shanghai, China), and used as received. Ammonium persulfate (APS), sodium dihydrogenphosphate dihydrate (NaH2PO4·2H2O), 3,5-dinitrosalicylic acid (DNS) and glutaraldehyde were obtained from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China) without further purification. Glucose from Macklin (Shanghai, China) was used after oven-drying to a constant weight at 90 ºC overnight. Fluorescein5(6)-isothiocyanate

(FITC),

N-Hydroxysuccinimide

(NHS)

and

N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) were purchased from Shanghai Macklin Biochemical Co., Ltd. PierceTM BCA protein assay kit was bought from Thermo Scientific (Shanghai, China). Microcrystalline cellulose was obtained from Sigma Aldrich (Shanghai, China) and carboxymethylcellulose sodium (CMC) was purchased from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China). Cellulase (blend of cellulases), a mixture of EG, CBH and β-G (from T. viride, CMCase activity of 15 U/mg), was bought from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China). β-G (from almonds, powder, a specific activity of ≥ 6 U/mg) 4


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was obtained from Sigma Aldrich (Shanghai, China). De-ionized (DI) water was used to prepare all aqueous solutions. Synthesis of a UCST-type copolymer PMAAc. The copolymer PMAAc was synthesized through the copolymerization reaction by two monomers of MAA and AAc, using ammonium persulfate as initiator and DI water as solvent respectively, and the specific procedures were as follows. First, 1.44 g of acrylic acid and 0.032 g of ammonium persulfate were dissolved in 10 mL of H2O, and then the mixed solution was transferred to a 3-neck round bottom flask at room temperature. Then, 0.42 g of methacrylamide dissolved in 10 mL of H2O was slowly added to the mixed solution under argon bubbling, over a period of 30 min at room temperature. The whole device was degassed by three freeze-pump-thaw cycles to ensure the reaction under vacuum and the reaction was then carried out at 60 ºC and 300 rpm with magnetic stirring for 4 h. After the reaction was finished, the resultant mixture was cooled to room temperature and was then centrifuged at 5 ºC and 6000 rpm for 2 min. The supernatant was removed and then the precipitate was poured out with following washing by DI water for three times. Finally, the product was dried in a vacuum oven overnight at 40 ºC to get the gelatin. The yield of the synthesis of PMAAc was more than 85%. Immobilization of cellulase onto PMAAc. The copolymer PMAAc was activated by glutaraldehyde and then it was covalently bounded with cellulase. 20 mg of PMAAc was dissolved with 1 mL of phosphate buffer solution (10 mM, pH 5.0) in 1.5 mL of EP tube in water bath at 50 ºC, and then different quantities of glutaraldehyde (10-60 µg) were added to activate the amino groups of PMAAc. The mixed solutions were transferred to a rocking incubator to react for 5 h at 50 ºC and 250 rpm. When the activated reaction was finished, the tubes were then centrifuged at 6000 rpm and 5 ºC for 5 min. The supernatant was removed and the precipitate was washed for three times, then the cleared precipitate was re-dissolved with 1 mL of phosphate buffer solution (10 mM, pH 4.0-7.0) containing different quantities of cellulase (1-7 mg). The obtained solutions were shaken at 35 ºC and 250 rpm in a rocking incubator for different times (1-6 h) and then was centrifuged at 8000 rpm 5



and 5 ºC for 10 min. Then the supernatant was removed and the precipitate was then washed by cooling DI water twice until there was no protein being detected in the washing solutions. The protein concentration of original, supernatant and washing solutions were respectively detected by the pierceTM BCA protein assay. The well handled immobilized cellulase was preserved at 4 ºC. Determination of the UCST of PMAAc and PMAAc-cellulase. The UCST of copolymer PMAAc and PMAAc-cellulase dissolved in 1 mL of phosphate buffer solution (10 mM, pH 5.0) was determined according to the reported cloud point method.27 The sample was firstly clarified in a glass tube equipped with a digital thermometer incubated in a thermostatic bath at 50 ºC, then the temperature was declined at a rate of 1 ºC per minute until the solution turned turbid. It was detected that the UCST of PMAAc and PMAAc-cellulase were 16 ºC and 19 ºC respectively. Determination of enzyme activity and concentration. The activity of both free and immobilized cellulase were determined by measuring the amount of the reducing sugar produced by the hydrolysis of 1% (w/v) CMC according the standard of the Commission on Biotechnology of the International Union of Pure and Applied Chemistry (IUPAC).28 The 4 mg of free or immobilized cellulase was dissolved with 100 µL of phosphate buffer solution (10 mM, pH 5.0) in a water bath at 50 ºC, then 900 µL of preheated 1% CMC in phosphate buffer (10 mM, pH 5.0) was respectively added to the cellulase solution to react for 10 min in a rocking incubator at 50 ºC. Subsequently, 50 µL of supernatant was sampled rapidly and mixed with 450 µL dinitrosalicylic acid (DNS) agent as a color indicator,29 and the mixture was then incubated in boiling water to conduct chromogenic reaction for 10 min. Then the EP tube was cooled in ice water once the reaction was over, and it was diluted to 1 mL. The reducing sugar produced was measured at 540 nm using a UV-vis spectrophotometer (UV-3600Plus; Shimadzu, Tokyo, Japan) at room temperature with glucose as a standard.30 The yield of glucose was calculated by the following equation:31

Yield of glucose (%) =

glucose (mg) ×100% substrate (mg) 6


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(1) One unit (U) of cellulase activity was defined by the amount of enzyme that produces 1.0 µmol of glucose from the substrate equivalent per minute at 50 °C and pH 5.0. Relative activity was defined as units of free and immobilized cellulase activity compared with the optimum. The cellulase content in solution was determined by the bicinchoninic acid method.32 When the immobilization reaction was finished, 20 µL of the supernatant and washing solution was respectively sampled with the addition of 400 µL prepared working reagent from PierceTM BCA protein assay kit in 1.5 mL of EP tubes in water bath at 37 ºC for 30 min. Then, all the tubes were cooled to the room temperature to be measured at 562 nm within 10 min. The protein concentration was determined by the BSA standard curve. The content of cellulase immobilized on PMAAc was determined from the initial free cellulase amount subtracting the cellulase amount in supernatant and washing solutions. Synthesis of fluorescently labeled cellulase (FITC-cellulase). 20 mg of cellulase was dissolved in 10 mL of phosphate buffer solution (10 mM, pH 5.0) under 4 ºC. EDC (10mg) and NHS (5mg) were added to the cellulase-containing solution to be uniformly mixed. Then, the solution was stirred for 1 h. Another 4 h stirring was conducted at 4 ºC in the dark after the addition of 100 µg of FITC to the above solution. The resultant solution was dialyzed to remove the remaining EDC, NHS and FITC in the FITC-cellulase containing solution (MWCO: 10k) for 24 h at 4 ºC in the dark. Catalytic activity of PMAAc-cellulase. The optimum pH and temperature of free and immobilized cellulase were determined by catalysis of CMC, which were respectively carried out over the pH range of 3.0-8.0 and temperature range of 30-80 °C for 10 min. The activities were measured by the DNS method described above. The results were then expressed as a percentage of relative activity. Stability and reusability of PMAAc-cellulase. The thermal stability experiment was carried out in phosphate buffer solution (10 mM, pH 5.0) in a rocking incubator at 70 °C for 5 h with a one-hour interval, and their pH stabilities were investigated in 7



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different phosphate buffer solutions (10 mM, pH 3.0-8.0) in a rocking incubator at 50 °C for 3 h. The storage stabilities of free and immobilized cellulase were measured in phosphate buffer solution (10 mM, pH 5.0) at 4 °C for 30 days with a five-day interval. In order to evaluate the reusability of the immobilized cellulase, the resultant biocatalyst was separated and washed with phosphate buffer solution (10 mM, pH 5.0) after each round and then the sample was resuspended for the next run. The hydrolysis reaction lasted 10 times and the enzyme activity of the first run was defined as 100 %. Hydrolysis of cellulose by immobilized cellulase with additional β-glucosidase (PMAAc-cellu&β-G). Based on the independent immobilization of cellulase, β-glucosidase was added to the cellulase solution as a supplemental enzyme to be co-immobilized on PMAAc. cellulase with various ratio (w/w) of β-glucosidase (10:1, 5:1, 3.3:1, 2.5:1, 2:1) were added to the PMAAc activated by 30 µg/mL of glutaraldehyde, then the mixed enzyme solution was shaken at 35 ºC and 250 rpm in a rocking incubator for 4 h. The resultant solution was centrifuged at 8000 rpm and 5 ºC for 10 min, and the product was then washed three times by cooled DI water. The co-immobilized PMAAc-cellu&β-G biocatalyst was stored at 4 ºC for use. The

hydrolysis

of

microcrystalline

cellulose

with

co-immobilized

PMAAc-cellu&β-G biocatalyst was then conducted. A set of mixtures of 1 mL of phosphate buffer solution (10 mM, pH 5.0) containing 0.1 mg of enzyme with different cellulase/β-glucosidase ratio (10:1, 5:1, 3.3:1, 2.5:1, 2:1, w/w), 20 mg of microcrystalline cellulose reacted in 1.5 mL EP tubes in a rocking incubator (250 rpm) at 50 °C for different times (4, 8, 12, 16, 20, 24h). After the hydrolysis reaction, the tubes were laid aside for a few minutes, and then the reducing sugar concentration was measured by DNS method. The reusability of the co-immobilized enzyme was investigated by the hydrolysis of insoluble microcrystalline cellulose into glucose for 8 cycles. 0.1 mg enzyme loading of PMAAc-cellu&β-G and 20 mg of microcrystalline cellulose were mixed with 1 mL of phosphate buffer solution (10 mM, pH 5.0) in a 1.5 mL EP tube, which was reacted in a rocking incubator (250 rpm) at 50 °C. After 24 h of reaction, the 8


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mixture was centrifuged at 6000 rpm and 35 °C (>UCST) for 5 min to remove the residual substrate. Then, the supernatant containing soluble immobilized cellulase with glucose was separated by centrifugation at 8000 rpm and 5 °C (UCST) and insoluble for separation at 5 ºC (

Recyclable soluble-insoluble UCST-type PMAAc ... - ACS Publications

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