Highly Efficient Synthesis of 2,5-Dihydroxypyridine using Pseudomonas sp. ZZ-5 Nicotine Hydroxylase Immobilized on Immobead 150

In this report, the use of immobilized nicotine hydroxylase from Pseudomonas sp. ZZ-5 (HSPHZZ) for the production of 2,5-dihydroxypyridine (2,5-DHP) from 6-hydroxy-3succinoylpyridine (HSP) in the presence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) is described. HSPHZZ was covalently immobilized on Immobead 150 (ImmHSPHZZ). ImmHSPHZZ (obtained with 5–30 mg of protein per gram of support) catalyzed the hydrolysis of HSP to 2,5-DHP. At a protein loading of 15 mg g−1, ImmHSPHZZ converted 93.6% of HSP to 2,5-DHP in 6 h. The activity of ImmHSPHZZ was compared with that of free HSPHZZ under various conditions, including pH, temperature, enzyme concentration, substrate concentration and stability over time, and kinetic parameters were measured. The results showed that ImmHSPHZZ performed better over wider ranges of pH and temperature when compared with that of HSPHZZ. The optimal concentrations of ImmHSPHZZ and substrate were 30 mg L−1 and 0.75 mM, respectively. Under optimal conditions, 94.5 mg L−1 of 2,5-DHP was produced after 30 min with 85.4% conversion. After 8 reaction cycles and 6 days of storage, 51.3% and 75.0% of the initial enzyme activity remained, respectively. The results provide a framework for development of commercially suitable immobilized enzymes that produce 2,5-DHP.


Introduction
Nicotine is a major pyridine alkaloid found in tobacco that causes smoking addiction and several diseases such as pulmonary disease and cancer [1,2].The manufacturing of tobacco products produces large amounts of toxic solid, liquid and airborne waste with high nicotine content [3,4].Nicotine is very toxic to human health and the disposal of tobacco waste is a serious ecological problem [5,6].As an environmentally friendly treatment, microbial degradation of nicotine is a promising approach because of its low cost and high efficiency.Several microorganisms including Arthrobacter species, Pseudomonas species, Agrobacterium tumefaciens S33, Ochrobactrum intermedium, Aspergillus oryzae, Rhodococcus species, Agrobacterium species and Sphingomonas species have been reported to degrade nicotine [7][8][9][10][11][12][13][14][15].Pyridine derivatives from nicotine are used extensively in functional materials and found in natural products that possess important biological activities.Recent studies have reported that several intermediates produced from nicotine degradation by microorganisms are important precursors (e.g., 2,5-dihydroxy-pyridine (2,5-DHP)) [5,16].In addition, the drug 5-aminolevulinic acid, used in cancer therapy, has been produced from degradation of nicotine by microorganisms [17].
Immobilization of enzymes is an essential facet of modern biotechnology.Enzyme immobilization (especially on magnetic carriers) offers many advantages when compared with soluble enzyme preparations, including the ability to improve the catalytic properties of the enzyme, easy enzyme recovery and reuse, and reduced catalytic processing costs [18][19][20].Various methods, including physical adsorption, covalent binding, cross-linking, entrapment and encapsulation, have been used for enzyme immobilization.Among them, the covalent binding method could make it difficult for the free enzyme to leach and exhibit high enzymatic activity due to the strong interaction between the enzyme and carrier function group [21][22][23].
In our previous work, the synthesis of 2,5-DHP from 6-hydroxy-3-succinoylpyridine (HSP) using HSP (6-hydroxy-3-succinoylpyridine) hydroxylase from Pseudomonas sp.ZZ-5 (HSPH ZZ ) was attempted, and the yield of 2,5-DHP reached 74.9% (w/w) in 40 min under optimal conditions [24].However, recovery and reuse of HSPH ZZ could not be achieved.Therefore, to improve the catalytic properties and reduce the cost of the biocatalytic process, HSPH ZZ should be immobilized.Until now, there are no published reports on the synthesis of 2,5-DHP by immobilized HSP hydroxylase.In this context, the aim of this work was to optimize the synthesis of 2,5-DHP catalyzed by HSPH ZZ covalently immobilized on Immobead 150 (ImmHSPH ZZ ) by determining the optimal pH, temperature, and enzyme and substrate concentrations.Kinetic parameters K m and k cat were determined and reusability was also examined.

Immobilization of Purified HSPH ZZ on Immobead 150
The immobilization of HSPH ZZ was performed with Immobead 150, and the immobilized HSPH ZZ was termed ImmHSPH ZZ .Protein loadings and immobilization times were 5-30 mg g −1 and 6 h, respectively.Table 1 shows that the immobilization efficiency of HSPH ZZ was 100% when the HSPH ZZ loading ranged from 5 mg g −1 to 20 mg g −1 .This result indicated that the support of Immobead 150 was not saturated at the high HSPH ZZ loading.The immobilization efficiency decreased to 45% when the HSPH ZZ loading increased to 30 mg g −1 .The retention of activity increased with increasing HSPH ZZ loading (5-15 mg g −1 ).The highest retention of activity of ImmHSPH ZZ was achieved (95%) at a HSPH ZZ loading of 15 mg g −1 .The retention of activity decreased to 45% when HSPH ZZ loading was further increased from 20 to 30 mg g −1 .The preparation of HSPH ZZ immobilized on Immobead 150 was used to catalyze the hydrolysis of HSP to 2,5-DHP with HSPH ZZ loadings of 5, 10, 15 and 20 mg g −1 (Figure 1).The reaction products catalyzed by ImmHSPH ZZ were identified using liquid chromatography-mass spectrometry (LC-MS.The reaction time was 6 h, and the yields of 2,5-DHP were 85.1%, 92.7%, 93.6% and 82.6% at HSPH ZZ loadings of 5, 10, 15 and 20 mg g −1 , respectively.These results demonstrated that the immobilized HSPH ZZ showed high hydroxylase activity after covalent binding to Immobead 150.

Scanning Electron Microscopy (SEM) Analysis of HSPH ZZ and ImmHSPH ZZ
The surface structure of HSPH ZZ before and after covalently immobilizing on Immobead 150 was observed using SEM (Figure 2).The hydroxylase bound to the surface of the beads and the surface morphology of HSPH ZZ immobilized to the beads differed when compared with that of the free enzyme.The ImmHSPH ZZ surface became spheroid in shape after immobilization, which may be caused by the formed covalent bond between an amino group located on the surface of the enzyme and the support matrix oxirane ring of Immobead 150 [25].Therefore, these results showed that nicotine hydroxylase HSPH ZZ was immobilized on Immobead 150.

Scanning Electron Microscopy (SEM) Analysis of HSPHZZ and ImmHSPHZZ
The surface structure of HSPHZZ before and after covalently immobilizing on Immobead 150 was observed using SEM (Figure 2).The hydroxylase bound to the surface of the beads and the surface morphology of HSPHZZ immobilized to the beads differed when compared with that of the free enzyme.The ImmHSPHZZ surface became spheroid in shape after immobilization, which may be caused by the formed covalent bond between an amino group located on the surface of the enzyme and the support matrix oxirane ring of Immobead 150 [25].Therefore, these results showed that nicotine hydroxylase HSPHZZ was immobilized on Immobead 150.

Scanning Electron Microscopy (SEM) Analysis of HSPHZZ and ImmHSPHZZ
The surface structure of HSPHZZ before and after covalently immobilizing on Immobead 150 was observed using SEM (Figure 2).The hydroxylase bound to the surface of the beads and the surface morphology of HSPHZZ immobilized to the beads differed when compared with that of the free enzyme.The ImmHSPHZZ surface became spheroid in shape after immobilization, which may be caused by the formed covalent bond between an amino group located on the surface of the enzyme and the support matrix oxirane ring of Immobead 150 [25].Therefore, these results showed that nicotine hydroxylase HSPHZZ was immobilized on Immobead 150.

Effect of pH on the Activity of HSPHZZ and ImmHSPHZZ
The effect of pH on the hydrolytic activity of HSPHZZ and ImmHSPHZZ was investigated at pH values between 5.0 and 10.0.As shown in Figure 3, free HSPHZZ showed higher hydrolytic activities over the pH range of 8.0-9.0 with a maximum activity at pH 8.5.After immobilization on Immobead 150, the optimum pH range was between 7.5 and 9.5, and maximal activity was observed at pH 9.0,

Effect of pH on the Activity of HSPH ZZ and ImmHSPH ZZ
The effect of pH on the hydrolytic activity of HSPH ZZ and ImmHSPH ZZ was investigated at pH values between 5.0 and 10.0.As shown in Figure 3, free HSPH ZZ showed higher hydrolytic activities over the pH range of 8.0-9.0 with a maximum activity at pH 8.5.After immobilization on Immobead 150, the optimum pH range was between 7.5 and 9.5, and maximal activity was observed at pH 9.0, indicating that the immobilized HSPH ZZ exhibited higher tolerance to alkaline pHs.ImmHSPH ZZ maintained over 50% of its maximum activity at pH 7.0-10.0,whereas the free HSPH ZZ maintained over 50% of its maximum activity at pH 7.0-9.0.These observations showed that immobilization of HSPHzz improved enzymatic performance over a wider pH range.This improved pH-stability may be because of the covalent bond formed and possible secondary interactions between the enzyme and the functional groups of the carrier, which enhance the stability of the molecular structure of the immobilized enzyme at various pH values [26].This behavior could be explained by influence of the partition effects on the enzymatic activities of the immobilized protein, which were from different concentrations of charged species in the micro-environment of the immobilized protein and reaction solution [27].
Catalysts 2018, 7, x FOR PEER REVIEW 4 of 11 indicating that the immobilized HSPHZZ exhibited higher tolerance to alkaline pHs.ImmHSPHZZ maintained over 50% of its maximum activity at pH 7.0-10.0,whereas the free HSPHZZ maintained over 50% of its maximum activity at pH 7.0-9.0.These observations showed that immobilization of HSPHzz improved enzymatic performance over a wider pH range.This improved pH-stability may be because of the covalent bond formed and possible secondary interactions between the enzyme and the functional groups of the carrier, which enhance the stability of the molecular structure of the immobilized enzyme at various pH values [26].This behavior could be explained by influence of the partition effects on the enzymatic activities of the immobilized protein, which were from different concentrations of charged species in the micro-environment of the immobilized protein and reaction solution [27].

Effect of Temperature on the Activity of HSPHZZ and ImmHSPHZZ
The effect of temperature on the hydrolytic activity of HSPHZZ and ImmHSPHZZ was evaluated.Immobilization of HSPHZZ strongly increased its thermostability when compared with that of the free HSPHZZ (Figure 4).Maximal activity of ImmHSPHZZ was observed at ~35 °C and more than half the maximal activity was observed at 15-45 °C.In contrast, the maximal activity of HSPHZZ was ~30 °C, and more than half the maximal activity was observed at 20-35 °C.Thermal stabilities of HSPHZZ and ImmHSPHZZ were investigated at 40 °C with increasing incubation periods up to 6 days.After incubation for 6 days, the enzymatic activity of ImmHSPHZZ was only partly reduced (~73%), whereas the activity of HSPHZZ had reduced to 13%.Thus, temperature has a lower impact on the activity profile of ImmHSPHZZ when compared with that of HSPHZZ.This result demonstrated that the immobilized HSPHZZ showed higher thermostability than the free enzyme [25,26].The thermostability of the immobilized HSPHZZ is in accordance with that of other previously reported immobilized enzymes.This improved thermostability may be because of the formation of the covalent linkage between HSPHZZ and Immobead 150, which changes the conformation of the enzyme as a result of the temperature changes [27].

Effect of Temperature on the Activity of HSPH ZZ and ImmHSPH ZZ
The effect of temperature on the hydrolytic activity of HSPH ZZ and ImmHSPH ZZ was evaluated.Immobilization of HSPH ZZ strongly increased its thermostability when compared with that of the free HSPH ZZ (Figure 4).Maximal activity of ImmHSPH ZZ was observed at ~35 • C and more than half the maximal activity was observed at 15-45 • C. In contrast, the maximal activity of HSPH ZZ was ~30 • C, and more than half the maximal activity was observed at 20-35 • C. Thermal stabilities of HSPH ZZ and ImmHSPH ZZ were investigated at 40 • C with increasing incubation periods up to 6 days.After incubation for 6 days, the enzymatic activity of ImmHSPH ZZ was only partly reduced (~73%), whereas the activity of HSPH ZZ had reduced to 13%.Thus, temperature has a lower impact on the activity profile of ImmHSPH ZZ when compared with that of HSPH ZZ .This result demonstrated that the immobilized HSPH ZZ showed higher thermostability than the free enzyme [25,26].The thermostability of the immobilized HSPH ZZ is in accordance with that of other previously reported immobilized enzymes.This improved thermostability may be because of the formation of the covalent linkage between HSPH ZZ and Immobead 150, which changes the conformation of the enzyme as a result of the temperature changes [27].

Effect of Enzymatic Concentration on the Activity of ImmHSPHZZ
Different enzymatic concentrations (10-50 mg mL -1 ) were studied to determine the optimal conditions for synthesis of 2,5-DHP.Interestingly, a lower yield of 2,5-DHP was obtained at higher concentrations of ImmHSPHZZ (40-50 mg mL −1 ; Figure 5).The highest 2,5-DHP yield (95.6%) was obtained at an enzyme concentration of 30 mg mL −1 .In general, the product yield increased as the concentration of the enzyme increased.However, the presence of excess catalyst (>30 mg mL −1 ) can result in enzyme agglomeration and diffusion problems, which can reduce reaction efficiency [28,29].An ImmHSPHZZ loading of 30 mg mL −1 offered the efficient hydrolysis of HSP and synthesis of 2,5-DHP.

Effect of Enzymatic Concentration on the Activity of ImmHSPH ZZ
Different enzymatic concentrations (10-50 mg mL -1 ) were studied to determine the optimal conditions for synthesis of 2,5-DHP.Interestingly, a lower yield of 2,5-DHP was obtained at higher concentrations of ImmHSPH ZZ (40-50 mg mL −1 ; Figure 5).The highest 2,5-DHP yield (95.6%) was obtained at an enzyme concentration of 30 mg mL −1 .In general, the product yield increased as the concentration of the enzyme increased.However, the presence of excess catalyst (>30 mg mL −1 ) can result in enzyme agglomeration and diffusion problems, which can reduce reaction efficiency [28,29].An ImmHSPH ZZ loading of 30 mg mL −1 offered the efficient hydrolysis of HSP and synthesis of 2,5-DHP.

Effect of HSP Concentration on the Activity of ImmHSPHZZ
The yields of 2,5-DHP produced by ImmHSPHZZ at different HSP concentrations (0.25-2.0 mM) are shown in Figure 6.The reaction rate increased with increasing HSP concentrations between 0.25 and 0.75 mM.The highest yield of 2,5-DHP was achieved at a HSP concentration of 0.75 mM.The yield of 2,5-DHP decreased when the HSP concentration was further increased from 1.0 to 2.0 mM.This may be because higher amounts of HSP limit substrate mass transfer to the active center of ImmHSPHZZ [30,31].The Michaelis-Menten equation was used for kinetic analysis of ImmHSPHZZ.The kinetic parameters of ImmHSPHZZ for HSP were calculated under optimal conditions and at the NADH concentration of 1.0 mM.As shown in Table 2, the kcat/Km value of ImmHSPHZZ (24.0 S −1 mM −1 ) toward HSP is higher than that of HSPHZZ (10.6 S −1 mM −1 ).Moreover, the kcat/Km value of ImmHSPHZZ (23.6 S −1 mM −1 ) toward NADH is higher than that of HSPHZZ (8.3 S −1 mM −1 ).Kinetic parameters (Km, kcat and Kcat/Km) for immobilized enzymes often differ when compared with the corresponding parameters obtained for the free enzyme [32,33].These results demonstrated that ImmHSPHZZ exhibited higher substrate affinity and catalytic efficiency when compared with that of HSPHZZ.

Effect of HSP Concentration on the Activity of ImmHSPH ZZ
The yields of 2,5-DHP produced by ImmHSPH ZZ at different HSP concentrations (0.25-2.0 mM) are shown in Figure 6.The reaction rate increased with increasing HSP concentrations between 0.25 and 0.75 mM.The highest yield of 2,5-DHP was achieved at a HSP concentration of 0.75 mM.The yield of 2,5-DHP decreased when the HSP concentration was further increased from 1.0 to 2.0 mM.This may be because higher amounts of HSP limit substrate mass transfer to the active center of ImmHSPH ZZ [30,31].The Michaelis-Menten equation was used for kinetic analysis of ImmHSPH ZZ .The kinetic parameters of ImmHSPH ZZ for HSP were calculated under optimal conditions and at the NADH concentration of 1.0 mM.As shown in Table 2, the k cat /K m value of ImmHSPH ZZ (24.0 S −1 mM −1 ) toward HSP is higher than that of HSPH ZZ (10.6 S −1 mM −1 ).Moreover, the k cat /K m value of ImmHSPH ZZ (23.6 S −1 mM −1 ) toward NADH is higher than that of HSPH ZZ (8.3 S −1 mM −1 ).Kinetic parameters (K m , k cat and K cat /K m ) for immobilized enzymes often differ when compared with the corresponding parameters obtained for the free enzyme [32,33].These results demonstrated that ImmHSPH ZZ exhibited higher substrate affinity and catalytic efficiency when compared with that of HSPH ZZ .

Effect of HSP Concentration on the Activity of ImmHSPHZZ
The yields of 2,5-DHP produced by ImmHSPHZZ at different HSP concentrations (0.25-2.0 mM) are shown in Figure 6.The reaction rate increased with increasing HSP concentrations between 0.25 and 0.75 mM.The highest yield of 2,5-DHP was achieved at a HSP concentration of 0.75 mM.The yield of 2,5-DHP decreased when the HSP concentration was further increased from 1.0 to 2.0 mM.This may be because higher amounts of HSP limit substrate mass transfer to the active center of ImmHSPHZZ [30,31].The Michaelis-Menten equation was used for kinetic analysis of ImmHSPHZZ.The kinetic parameters of ImmHSPHZZ for HSP were calculated under optimal conditions and at the NADH concentration of 1.0 mM.As shown in Table 2, the kcat/Km value of ImmHSPHZZ (24.0 S −1 mM −1 ) toward HSP is higher than that of HSPHZZ (10.6 S −1 mM −1 ).Moreover, the kcat/Km value of ImmHSPHZZ (23.6 S −1 mM −1 ) toward NADH is higher than that of HSPHZZ (8.3 S −1 mM −1 ).Kinetic parameters (Km, kcat and Kcat/Km) for immobilized enzymes often differ when compared with the corresponding parameters obtained for the free enzyme [32,33].These results demonstrated that ImmHSPHZZ exhibited higher substrate affinity and catalytic efficiency when compared with that of HSPHZZ.The production of 2,5-DHP from HSP by ImmHSPH ZZ was measured under optimal conditions (at 35 • C and pH 9.0 in 20 mM Tris-HCl buffer) with ImmHSPH ZZ at a concentration of 30 mg mL −1 .The immobilized enzyme produced 2,5-DHP (97.2 mg L −1 ) at a conversion of 85.4% (w/w) after a reaction time of 30 min (Figure 7).In our previous work, the yield of 2,5-DHP using free HSPH ZZ reached 74.9% in 40 min under optimal conditions [24].Therefore, the yield of 2,5-DHP catalyzed by ImmHSPH ZZ is higher when compared with that of HSPH ZZ , and 2,5-DHP was produced in a shorter reaction time (30 min) when HSP was catalyzed by ImmHSPH ZZ .The production of 2,5-DHP from HSP by ImmHSPH ZZ was also higher than that of the nicotine HSP hydroxylase from A. tumefaciens S33 (a conversion of 69.7% in 50 min) [10].These results demonstrated that enzymatic transformation of HSP to 2,5-DHP by ImmHSPH ZZ was superior to free HSPH ZZ and likely to be suitable for commercial applications.The production of 2,5-DHP from HSP by ImmHSPHZZ was measured under optimal conditions (at 35 °C and pH 9.0 in 20 mM Tris-HCl buffer) with ImmHSPHZZ at a concentration of 30 mg mL −1 .The immobilized enzyme produced 2,5-DHP (97.2 mg L −1 ) at a conversion of 85.4% (w/w) after a reaction time of 30 min (Figure 7).In our previous work, the yield of 2,5-DHP using free HSPHZZ reached 74.9% in 40 min under optimal conditions [24].Therefore, the yield of 2,5-DHP catalyzed by ImmHSPHZZ is higher when compared with that of HSPHZZ, and 2,5-DHP was produced in a shorter reaction time (30 min) when HSP was catalyzed by ImmHSPHZZ.The production of 2,5-DHP from HSP by ImmHSPHZZ was also higher than that of the nicotine HSP hydroxylase from A. tumefaciens S33 (a conversion of 69.7% in 50 min) [10].These results demonstrated that enzymatic transformation of HSP to 2,5-DHP by ImmHSPHZZ was superior to free HSPHZZ and likely to be suitable for commercial applications.

Stability of Storage and Reusability
The stability of storage and reusability is an important requirement for industrial enzyme applications.Immobilization of the enzyme may improve resistance to reaction conditions and conformational changes, which reduces the likelihood of denaturation [34,35].The storage stability and operational stability of ImmHSPHZZ towards HSP were evaluated (Figure 8).The results showed that the immobilized enzyme kept 51.3% of its activity up until eight cycles.These results may be explained that HSPHZZ release from the support during recycle use due to weak interaction between Immobead 150 and the enzyme.To investigate the storage stability of ImmHSPHZZ, the activity of HSPHZZ after incubating the enzyme at 4 °C for different time intervals was measured.After 6 days, the relative activity of ImmHSPHZZ remained greater than 75.0% of the initial activity, whereas that

Stability of Storage and Reusability
The stability of storage and reusability is an important requirement for industrial enzyme applications.Immobilization of the enzyme may improve resistance to reaction conditions and conformational changes, which reduces the likelihood of denaturation [34,35].The storage stability and operational stability of ImmHSPH ZZ towards HSP were evaluated (Figure 8).The results showed that the immobilized enzyme kept 51.3% of its activity up until eight cycles.These results may be explained that HSPH ZZ release from the support during recycle use due to weak interaction between Immobead 150 and the enzyme.To investigate the storage stability of ImmHSPH ZZ , the activity of HSPH ZZ after incubating the enzyme at 4 • C for different time intervals was measured.After 6 days, the relative activity of ImmHSPH ZZ remained greater than 75.0% of the initial activity, whereas that of HSPH ZZ was only ~9% of the initial activity.These results indicated that the storage stability of ImmHSPH ZZ was clearly superior when compared with that of HSPH ZZ .
Catalysts 2018, 7, x FOR PEER REVIEW 8 of 11 of HSPHZZ was only ~9% of the initial activity.These results indicated that the storage stability of ImmHSPHZZ was clearly superior when compared with that of HSPHZZ.

Materials
2,5-DHP was purchased from SynChem OHG (Altenburg, Germany).The Immobead 150 support and succinic acid were purchased from Sigma Aldrich Co. (St.Louis, MO, USA).HSP was obtained from Professor Hongzhi Tang at Shanghai Jiao Tong University.All other chemicals used were analytical grade quality and obtained from commercial sources in China.

Materials
2,5-DHP was purchased from SynChem OHG (Altenburg, Germany).The Immobead 150 support and succinic acid were purchased from Sigma Aldrich Co. (St.Louis, MO, USA).HSP was obtained from Professor Hongzhi Tang at Shanghai Jiao Tong University.All other chemicals used were analytical grade quality and obtained from commercial sources in China.

Multipoint Immobilization of HSPH ZZ on Immobead 150
The support of Immobead 150 (0.2 g) was added to a HSPH ZZ solution (5 mL) in 20 mM sodium bicarbonate buffer (pH 8.5), with HSPH ZZ loadings ranging from 5 mg g −1 to 30 mg g −1 .The mixture was incubated at 4 • C for 6 h in an orbital shaker operating at 150× rpm.To check for covalent binding, the mixture was washed with 1 M NaCl and ethylene glycol (30%, v/v), centrifuged, vacuum freeze-dried and stored at 10 • C. The immobilization efficiency (IE, %) and retention of activity (R, %) were calculated according to Madalozzo et al. [36].

Characterization of HSPH ZZ and ImmHSPH ZZ
The activity of HSPH zz and ImmHSPH ZZ hydroxylase was determined by the catalysis of HSP to 2,5-DHP, as described previously [24].The production of 2,5-DHP was measured by an electron spray ionization (ESI) source on an AB Sciex Triple Quad 5500 mass spectrometer (AB Sciex, Framingham, MA, USA) with an Agilent 1290 infinity liquid chromatography (LC) system for ultra high performance liquid chromatography (UHPLC).The mobile phase was methanol/acetic acid (v/v, 25:75) at a flow rate of 0.5 mL min −1 .One unit of HSPH ZZ and ImmHSPH ZZ were defined as the amount of enzyme required to synthesize 1 mol 2,5-DHP per min under the standard conditions.
The activities of HSPH ZZ and ImmHSPH ZZ were determined as a function of pH (5.0-10.0),temperature (5-50 • C), enzymatic concentration (10-50 mg mL −1 ) and substrate (HSP) concentration (0.25-2.0 mM).The enzymatic activities of the free and immobilized enzyme were determined using HSP at concentrations ranging from 0.01 to 1 mM.The corresponding K m and k cat values were calculated using Hanes-Wolff plots and the Michaelis-Menten equation.

Stability of ImmHSPH ZZ
The reusability of ImmHSPH ZZ was examined by the hydrolytic activity assay under optimal conditions.At the end of each cycle, the separation of ImmHSPH ZZ from the reaction mixture was achieved by filtration and washed with 1 mL of 20 mM bicarbonate buffer (pH 8.5).Recovered ImmHSPH ZZ was then dried at room temperature for 24 h before use in the next cycle.
Relative activity was calculated by defining the first reaction as 100%.

SEM Assay of ImmHSPH ZZ
The ImmHSPH ZZ was sputtered with gold and the structures were observed using a Quanta-200 scanning electron microscope (FEI, Amsterdam, Holland).

Statistical Analysis
Triplicate experiments were performed for each parameter investigated and the mean and standard deviation values were reported.

Conclusions
In this study, nicotine hydroxylase from Pseudomonas sp.ZZ-5 was immobilized on Immobead 150 using covalent binding methods to optimize enzymatic production of 2,5-DHP from HSP.This preparation was characterized by SEM techniques to characterize the morphology of the immobilized enzyme on the support and the enzyme-support interaction.ImmHSPH ZZ displayed higher hydrolysis activity and catalytic performance than free HSPH ZZ , and exhibited better thermal stability, storage stability, and reusability when compared with the free enzyme.Under optimal conditions, ImmHSPH ZZ produced 94.5 mg L −1 of 2,5-DHP from 200 mg L −1 of HSP after 30 min with 85.4% conversion.These results demonstrated that immobilized HSPH ZZ is a prospective system for the enzymatic production of 2,5-DHP in biotechnology applications.

Figure 4 .
Figure 4. Temperature optima (a) and thermostability (b) of HSPHZZ and ImmHSPHZZ.(a) Temperature optimums of HSPHZZ and ImmHSPHZZ were determined with HSP as substrate in 20 mM Tris-HCl buffer (pH 9.0) at temperatures ranging from 5 to 50 °C.(b) Thermostability of HSPHZZ and ImmHSPHZZ.The residual enzyme activity was measured after incubation of the enzyme at 40 °C.

Figure 4 .
Figure 4. Temperature optima (a) and thermostability (b) of HSPH ZZ and ImmHSPH ZZ .(a) Temperature optimums of HSPH ZZ and ImmHSPH ZZ were determined with HSP as substrate in 20 mM Tris-HCl buffer (pH 9.0) at temperatures ranging from 5 to 50 • C. (b) Thermostability of HSPH ZZ and ImmHSPH ZZ .The residual enzyme activity was measured after incubation of the enzyme at 40 • C.

Figure 5 .
Figure 5.Effect of enzyme concentration of ImmHSPHZZ on the production of 2,5-DHP.

Figure 6 .
Figure 6.Effect of substrate concentration on the production of 2,5-DHP for HSPHZZ and ImmHSPHZZ.

Figure 5 .
Figure 5.Effect of enzyme concentration of ImmHSPH ZZ on the production of 2,5-DHP.

Figure 5 .
Figure 5.Effect of enzyme concentration of ImmHSPHZZ on the production of 2,5-DHP.

Figure 6 .
Figure 6.Effect of substrate concentration on the production of 2,5-DHP for HSPHZZ and ImmHSPHZZ.

Figure 6 .
Figure 6.Effect of substrate concentration on the production of 2,5-DHP for HSPH ZZ and ImmHSPH ZZ .

Table 1 .
Effect of the protein to support ratio (protein loading) on the immobilization of purified HSPHZZ on Immobead 150.

Table 2 .
Kinetic parameters of HSPHZZ and ImmHSPHZZ for the substrate of HSP or DADH.

Table 2 .
Kinetic parameters of HSPHZZ and ImmHSPHZZ for the substrate of HSP or DADH.